BIBLIOGRAPHY MATIW, AGATE VICTRICIA P....
BIBLIOGRAPHY
MATIW, AGATE VICTRICIA P. MARCH 2013. Microbial Antagonists (MA’s)
and Organic-based Products as Potential Biopesticides against Black Leaf Spot
(Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa). Benguet State
University, La Trinidad, Benguet.
Adviser: Luciana M. Villanueva, Ph.D.
ABSTRACT
The study aimed to; 1. investigate the in-vitro activity of ten (10) microbial
antagonists against X. campestris pv. vitians, 2. determine the effect of each microbial
antagonists on the germination and seedling vigor of lettuce seedlings, 3. investigate the
in-vitro activity of five (5) aqueous plant extracts with three (3) different extracting
solvents at different concentrations against X. campestris pv. vitians, and 4. identify the
phytochemicals present in the extracts using different extracting solvents.
Results of the in-vitro activity of ten bacterial antagonists against X. campestris pv.
vitians revealed the significant inhibitory effect of PCN-2011-004 on the growth of the
pathogen. This was comparable to the standard biofungicide, Virtuoso and Isolate 158.
Although lower in efficacy, PCN-2011-002 and PCN-2011-005 also effected a growth
suppression of X. campestris pv. vitians.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
The highest germination was noted in Isolate 131. However, this was comparable
to all the microbial antagonists tested and the standard treatments. The lowest percentage
germination was obtained from the untreated seeds.
The highest mean height was noted in STR-2011-001 treated seeds. Nevertheless,
this did not significantly differ from Virtuoso, Isolate 131, Isolate 94, Isolate 158, PCN-
2011-003 and PCN-2011-005. On the other hand, Isolate 73, PCN-2011-002, PCN-2011-
004 and Isolate 31 gave significantly shorter seedlings than the above microbial antagonists
but differed significantly from sterile distilled water treated seeds.
The main effect of plant extracts on the growth of X. campestris pv. vitians was not
significant. Significant differences were however observed on the different extractants.
Vinegar was the most effective but it did not significantly differ from wine. The least
effective was water. On the other hand, the highest concentration (1:1) showed
significantly the highest inhibition zone. This was comparable to 1:5 and 1:10
concentrations but was significantly different from 1:15. No significant interaction was
noted among the factors evaluated.
The phytochemical analysis revealed that alkaloids, diterpenes, triterpenes and
flavonoids can be extracted by water. Triterpenes, diterpenes and flavonoids can be
extracted by wine. On the other hand, diterpenes, triterpenes, phenolics and flavonoids can
be extracted by vinegar.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
RESULTS AND DISCUSSION
Sensitivity Test of X. campestris pv. vitians to different microbial antagonists (MA’s)
Among the microbial antagonists tested, PCN-2011-004 gave the highest inhibition
zone followed by Isolate 158 with inhibition means of 4 and 3.33 mm, respectively (Table
1). The two microbial agents were comparable to Virtuoso, the standard treatment, with a
mean of 4.67 mm. Meanwhile, PCN-2011-005 with inhibition mean of 3.03 mm was
similar to PCN-2011-004, Isolate 158 and PCN-2011-002. No significant differences were
observed among the following treatments: sterile distilled water, Isolate 73, STR-2011-
001, Isolate 94, Isolate 31, Isolate 131, PCN-2011-003 and cuprous oxide as they did not
actively inhibit the growth of the pathogen. The two isolates comparable to Virtuoso can
be manipulated to either equal the antibiotic potency of Virtuoso or to overcome the latter’s
diffusion rate, in-vitro.
Antibiosis, the antagonism resulting from the production by one microorganism
Table 1. Efficacy of the different microbial antagonists, Virtuoso and cuprous oxide on the
growth of X. campestris pv. vitians
Treatment
Actual Mean
Transformed Mean
Sterile Distilled water
0.00d
0.71d
PCN-2011-004(Bacillus sp.)
4.00ab
4.00ab
PCN-2011-005 (Bacillus sp.)
2.33c
2.33c
Isolate 73 (B. pumilus)
0.00d
0.71d
PCN-2011-002 (Bacillus sp.)
3.03bc
3.03bc
STR-2011-001 (Bacillus sp.)
0.00d
0.71d
Isolate 94 (Flavobacterium sp.) 0.00d
0.71d
Isolate 31 (Bacillus sp.)
0.00d
0.71d
Isolate 131 (B. pumilus)
0.00d
0.71d
Isolate 158 (Pseudomonas sp.)
3.33abc
3.33abc
PCN-2011-003 (Bacillus sp.)
0.00d
0.71d
Virtuoso (B. subtilis)
4.67a
4.33a
Cuprous oxide
0.00d
0.71d
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
of secondary metabolites toxic to other microorganisms, is a form of microbial antagonism
wherein direct interaction between two microorganisms shares the same ecological niche
(Alabouvette et al., 2006). This derived mechanism accounts for the formation of clear
zones between the pathogen and the BCA’s. Further, Alabouvette et al stated that antibiosis
is a very common phenomenon responsible for the activity of many BCA’s such as
fluorescent Pseudomonas and Bacillus spp.
Fluorescent pseudomonads owe their fluorescence to an extracellular diffusible
pigment called pyoverdin (Pvd) or pseudobactin. This pigment has high affinity for Fe3+
ions and is a siderophore (iron-carrier) of the producer strain (Meyer and Abdallah, 1978).
In iron depleted media in vitro, Pvd-producing Pseudomonas spp. inhibited the growth of
bacteria with less potent siderophores (Kloepper et al., 1980), explaining the small halo
produced around the susceptible pathogen. Under certain conditions therefore, Pvd
functions as a diffusible bacteriostatic antibiotic.
The mechanism for growth suppression employed by Bacillus spp. against X.
campestris pv. vitians as observed was the production of antibiotics, although the type of
antibiotics produced in this case is not known. In fungal diseases, B. subtilis produced
mycosubtilin against Pythium aphanidermatum (Leclere et al., 2005) while B.
amyloliquefaciens produced bacillomycin and fengycin against Fusarium oxysporum
(Koumoutsi et al., 2004). Purification and identification therefore of the toxic metabolites
produced by the Bacillus species are necessary to understand fully the mechanism of
control expressed in vitro to be able to modify its structure and improve its durability as
this antibiotic expression can be lost over time.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
Germination rate, seedling height and vigor index as affected by the different microbial
antagonists
Isolate 131 gave the highest germination rate at 97.22% with significant differences
to Isolate 158, PCN-2011-003, PCN-2011-005, and PCN-2011-002 at 95.83%, 95.83%,
95.55%, and 91.67% respectively (Table 2). However, the latter isolates were comparable
to Isolate 31, STR-2011-001, Isolate 94, Isolate 73, Virtuoso, Cuprous oxide and PCN-
2011-004 at 90.27%, 90%, 88.87%, 86.11%, 83.34%, 81.94% and 81.94%. Further, the
latter isolates are not significantly different to the control.
Germination test on lettuce variety Tyrol revealed that Virtuoso stimulated
germination at 90% while cuprous oxide at 89.25% (Calderon, 2011). Results of which
were higher compared to what was obtained in this experiment. Since variables can be
manipulated, it can be inferred that the percentage germination expressed by the standard
treatments operate on numerical variables as 80-90% germination capacity.
Table 2. Influence of microbial antagonists on the germination and growth of lettuce
seedlings
Seedling
Seedling
Seedling
TREATMENTS
Germination
Height
Vigor Index
Percentage
(mm)
(%)
Sterile Distilled water
75.00b
67.07d
5033.40d
PCN-2011-004(Bacillus sp.)
81.94ab
82.73bcd
6809.38bcd
PCN-2011-005 (Bacillus sp.)
95.55a
90.80abc
8670.02ab
Isolate 73 (B. pumilus)
86.11ab
83.13bcd
7178.80abcd
PCN-2011-002 (Bacillus sp.)
91.67a
83.13bcd
7630.28abc
STR-2011-001 (Bacillus sp.)
90.00ab
102.40a
9219.50a
Isolate 94 (Flavobacterium sp.)
88.89ab
88.33abc
7856.47abc
Isolate 31 (Bacillus sp.)
90.28ab
82.53bcd
7449.39ab
Isolate 131 (B. pumilus)
97.22a
92.33ab
8929.56ab
Isolate 158 (Pseudomonas sp.)
95.83a
85.80abcd
8248.61ab
PCN-2011-003 (Bacillus sp.)
95.83a
85.80abcd
7838.89abc
Virtuoso (B. subtilis)
83.34ab
86.33abc
7166.49abcd
Cuprous oxide
81.94ab
72.87cd
5960.53cd
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
The ability of cuprous oxide to induce germination rate could be attributed to the
presence of growth promoting hormones such as IAA, auxins, cytokinins, and gibberellins
added to its over-all ingredient. Calderon (2011) citing an article from the agribusiness
week stated that copper is an essential microelement needed by plants to promote seed
production and formation. Maneb, for example, induces germination of seeds due to the
presence of Zn as one of its ingredients. BCA’s also produce Indole Acteic Acid (IAA),
auxins, cytokinins and gibberellins to promote growth. When Ahmad, et al (2006) screened
free-living rhizospheric bacteria for their multiple plant growth promoting activities,
Pseudomonas and Bacillus produced IAA, along with Azotobacter isolates.
In the determination of the effect of the biocontrol agents on the seedling height of
lettuce, STR-2011-001 promoted seedling growth at 102.40 mm, being the highest among
the bacterial agents, followed by Isolate 131, PCN-2011-005, Isolate 94, Virtuoso, Isolate
158, PCN-2011-003, Isolate 73, PCN-2011-004, Isolate 31, cuprous oxide and distilled
water at 92.33, 90.80, 88.33, 86.33, 85.80, 85.79, 83.13, 83.13, 82.73, 82.53, 72.87 and
67.07 mm, respectively.
Statistically, STR-2011-001 was comparable to Isolate 131, PCN-2011-005, Isolate
94, Virtuoso, Isolate 158, and PCN-2011-003 and significantly different to PCN-2011-002,
Isolate 73, PCN-2011-004, Isolate 31, cuprous oxide, and the control. These reactions
indicate that PGPR capacity of microbial agents does not necessitate that germination
stimulation and growth promotions are closely linked characteristics. One biocontrol agent
may stimulate germination; one may promote growth, while one may have both. Isolate
131 is an example.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
In the seedling vigor index tests, STR-2011-001 gave the highest index at 9219.50,
however it was comparable to Isolate 131, PCN-2011-005, Isolate 158, Isolate 94, PCN-
2011-003, PCN-2011-002, Isolate 31, Isolate 73, and Virtuoso at 8929.56, 8670.02,
8248.61, 7856.47, 7838.89, 7630.28, 7449.39, 7178.80, 7166.49 and differed significantly
from PCN-2011-004, cuprous oxide and the control with respective indexes of 6809.38,
5960.53 and 5033.40.
Note that Pseudomonas sp. (Isolate 158) gave an index of 8248.60. In the sensitivity
screening, its antibiotic behavior was linked to its production of siderophores. Schroth et
al (1982) postulated that the resulting siderophore hypothesis exert their plant growth
promoting activity by depriving pathogens of iron. An example cited by Haas and Defago
(2005) was the suppression of Fusarium wilt and take-all, by P. putida strain B10 but this
suppression was lost when the soil was amended with Iron; a move which repressed
siderophore production in this strain.
To follow, PGPR can also protect a crop against phyto diseases. Harman et al
(2004) stated that some biocontrol PGPR elicit a phenomenon known as Induced Systemic
Resistance (ISR) in the host plant. ISR is an indirect phenomenon expressed by any plant
in reaction to chemical molecules from natural or synthetic origins (Alabouvette et al.,
2005) which were assumed to be produced by the Bacillus and Pseudomonas strains
utilized in this study. For example, in Pseudomonas strains, a combination of pyocyanin
and pyochelin seems to be most effective for inducing resistance in tomato (Audenaert et
al., 2002). The plant growth stimulating volatile 2,3-butanediol that is found in Bacillus
spp. can also initiate ISR (Ryu et al., 2004).
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
Calderon (2011) revealed that bacterial isolate 9 induced germination at 95.5%,
comparable to Virtuoso and Trichoderma with treatment means of 91.5% and 90%
respectively. It also promoted seedling height with means of 48.60 mm although not
comparable to Trichoderma and Virtuoso with treatment means of 61.56 mm and 54.31
mm respectively. Interestingly, bacterial isolate 9 reduced black leaf spot severity with a
rating of 1.92, in comparison to the control (3.08) following Virtuoso at 1.83 rating. This
could illustrate the effect of PGPR on ISR of lettuce against X. campestris pv. vitians.
However, it should be noted that ISR and other modes of action are not mutually exclusive;
ISR might only exert a complementary effect to microbial antagonism (Alabouvette et al.,
2005).
Collectively, the results show two promising biological control agents with the
capacity to suppress growth of X. campestris pv. vitians in-vitro, stimulate germination and
promote seedling growth. These are PCN-2011-005 and Isolate158 as shown by table 3 in
comparison to the other standard treatments.
Statistically, Virtuoso produced the highest mean of inhibition at 4.33 and was
significantly different from Isolate 158, PCN-2011-005, cuprous oxide and the control.
Isolate 158 and PCN-2011-005 were however not significantly different from each other.
Table 3. Efficacy of promising microbial antagonists on the growth of X. campestris
pv. vitians and on the germination, seedling height and seedling vigor of lettuce
TREATMENTS
Inhibition %
Seedling
Seedling
Zone
germination height
vigor
(mm)
(mm)
index
T0 – SDW
0.00c
75.00b
67.07c
5033.40c
T2 – PCN-2011-005 (Bacillus sp.) 2.33b
95.55a
90.80a
8670.02a
T3 – Isolate 158 (Pseudomonas
3.33b
95.83a
85.80ab
8248.61ab
sp.)
T4– Virtuoso (B. subtilis)
4.67a
83.34ab
86.33ab
7166.49abc
T5 – Cuprous oxide
0.00c
81.94ab
72.87bc
5960.53bc
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
In terms of percentage germination, Isolate 158 was the highest at 95.83. This was
however not significantly different from PCN-2011-005. Both treatments were however
comparable to Virtuoso and cuprous oxide. In terms of seedling height, PCN-2011-005
produced the highest mean at 90.80, comparable to Isolate 158 and Virtuoso but
significantly different from distilled water and cuprous oxide. Cuprous oxide was however
comparable to Isolate 158 and Virtuoso. In terms of seedling vigor, PCN-2011-005
produced the highest mean at 8670.02, comparable to Isolate 158 and Virtuoso but
significantly different from distilled water and cuprous oxide. Cuprous oxide was however
comparable to Isolate 158 and Virtuoso.
Note however, that many effective biocontrol PGPR elicit ISR without necessarily
producing antibiotics (Zhender et al., 2001; Ongena et al., 2004; and Ton et al., 2002).
This could be true to both STR 1 and Isolate 131.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
a
b
c
d
e
f
g
h
i
j
k
l
m
Fig 1. Effect of microbial antagonists on the height of lettuce seedlings: a. Sterile
Distilled Water; b. PCN-2011-004; c. PCN-2011-005; d. Isolate 73; e. PCN-2011-
002; f. STR-2011-001; g. Isolate 94; h. Isolate 31; i. Isolate 131; j. Isolate 158; k.
PCN-2011-003; l. Virtuoso; m. cuprous oxide.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
Sensitivity screening of X. campestris pv. vitians to different organic-based products at
different concentrations
Extract
a
a
12
a
10
a
a
8
6
Extract
4
2
0
Piper betle
Pandanus
Allium
Euphorbia
Psidium
sp.
sativum
sp.
guajava
Fig 2. Efficacy of extracts against X. campestris pv. vitians
Statistically, no significant difference was obtained from the means of the data
analyzed, but numerically, A. sativum was the best extract at 10.21, followed by P. guajava,
Euphorbia sp., P. betle, and Pandanus sp., at 9.22, 7.44, 6.56, and 5.52 respectively. The
greater antimicrobial activity of A. sativum could be attributed to the compound allicin, one
of the active principles of freshly crushed garlic homogenates. Allicin has a variety of broad
spectrum antimicrobial activities. In its pure form, it was found to inhibit Gram negative
bacteria causing human diseases (Ankri and Mirelman, 1999). Lirio et al (1999) reported
that garlic was able to inhibit the growth of X. campetsris pv. campestris, in-vitro. The
main antimicrobial effect of allicin is due to its chemical reaction with thiol groups of
various enzymes, e.g. alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase
which can affect essential metabolism of cysteine proteinase activity involved in the
virulence of some bacteria (Ankri and Mirelman, 1999). Its effect on X. campestris pv.
vitians is one concern raised during this study; although, Cavallito and Bailey (1945)
reported that inhibition of thiol-containing enzymes in the microorganism by the rapid
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
reaction of the thiosulfanates with thiol groups was assumed to be the main mechanism
involved in the antibiotic effect. It is reasonable to conclude, therefore, that the wide
spectrum antimicrobial effects of allicin are due to the multiple inhibitory effects they may
have on various thiol-dependent enzymatic systems (Ankri and Mirelman, 1999).
Berdy et al 1982 and Caceres et al 2005 attributed the antibiotic effect of P. guajava
to 2 compounds, guajaverin and psidiolic acid against bacteria. Agarwal et al (2012)
postulated that the antibacterial activity of leaves of P. betle against gram positive and gram
negative bacteria is due to the presence of metabolic toxins or broad spectrum antimicrobial
compounds. Euphorbia sp. and Pandanus sp. also have antibiotic compounds responsible
for the inhibition zones formed around the agar wells against X. campestris pv. vitians.
Generally, plants elicit a certain chemical as a form of defense mechanism against an
invasion by a foreign material.
Extractant
a
20
15
ab
Extractant
10
b
5
0
Water
Wine
Vinegar
Fig 3. Efficacy of water, wine and vinegar as organic solvents to extract phytochemical
compounds from the plant materials
Vinegar showed the highest mean at 15.07, followed by wine and water with means
of 6.57 and 1.73, respectively (Fig. 3). Statistically, vinegar was the most effective but this
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
was comparable to wine and differed significantly with water. Vinegar contains acetic acid,
a form of ethanol responsible for the alcohol content of the vinegar. Wines in particular
may contain either ethanol or methanol. Alcohols are good solvents for the extraction of
phytochemicals situated within the plant cells as they are more efficient in cell wall
degradation causing polyphenols to be released from cells. The decrease in activity of
aqueous extract (water) can be ascribed to the enzyme polyphenol oxidase, which degrade
polyphenols in water extracts whereas in methanol and ethanol (alcohols) they are inactive
(Lapornik et al., 2005). Reasons as to why wine is lower than vinegar, although they have
alcohols, will be discussed further in the phytochemical analysis.
Fig 4 illustrates the effect of the concentration on the efficacy of the extracts.
Concentration
a
20
15
ab
10
Concentration
b
ab
5
0
1:01
1:05
1:10
1:15
1:1
1:5
Fig 4. Effect of concentrations on the efficacy of plant extracts against X. campestris pv.
vitians
Apparently, concentration 1:1 was the most effective but comparable to 1:5, 1: 10
and 1:15 with respective means of 15.14, 6.93, 4.80 and 4.29. The inference therefore is
that, as the dilution of solvents to the plant materials is increased, the efficacy of the
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
concentrations against X. campestris pv. vitians decreases. Vis-à-vis, as the dilution
decreases, the efficacy of the concentrations increases.
On the other hand, no significant interaction was observed among the three factors,
namely: extract, extractant and concentration.
A
1:1
1:5
1:10
1:15
B
1:1
1:5
1:10
1:15
C
1:1
1:5
1:10
1:15
Fig 5. Effect of different extracting solvents and concentrations on the efficacy of
P. betle against X. campestris pv. vitians: A. Water, B. Wine, C. Vinegar
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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MATIW, AGATE VICTRICIA P. MARCH 2013
A
1:1
1:5
1:10
1:15
B
1:1
1:5
1:10
1:15
C
1:1
1:5
1:10
1:15
Fig 6. Effect of different extracting solvents and concentrations on the efficacy of
Pandanus sp. against X. campestris pv. vitians; A. Water, B. Wine. C. Vinegar
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
A
1:1
1:5
1:10
1:15
A
1:1
1:5
1:10
1:15
A
1:1
1:5
1:10
1:5
Fig 7. Effect of different extracting solvents and concentrations on the efficacy of
l
A. sativum against X. campestris pv. vitians; A. Water, B. Wine, C. Vinegar
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
A
1:1
1:5
1:10
1:15
B
1:1
1:5
1:10
1:15
C
1:1
1:5
1:10
1:15
Fig 8. Effect of different extracting solvents and concentrations on the efficacy of
l
Euphorbia sp. against X. campestris pv. vitians; A. Water, B. Wine, C. Vinegar
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
A
1:1
1:5
1:10
1:15
A
1:1
1:5
1:10
1:15
A
1:1
1:5
1:10
1:15
Fig 9. Effect of different extracting solvents and concentrations on the efficacy of
P. guajava against X. campestris pv. vitians; A. Water, B. Wine, C. Vinegar
l
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
Phytochemical analysis of the different plant extract - extractant combination
Extensive research has been performed worldwide and important evidences were
collected to show the immense potential of plants used in various traditional therapeutic
systems (Caunii et al., 2005). Many scientists nowadays are exploiting this complex line
of systems to answer the problems on resistance exhibited by pathogens to the existing
antibiotics. The exploitation of the botanical world begins with the understanding of the
phytochemicals present in the plant with specific concern to the bioactive compounds in
the plant materials tested against X. campestris pv. vitians.
The phenolic compounds present in extracts of plants are always a mixture of
different classes of phenols selectively soluble in the solvents (Caunii et al., 2005).
Successful determination of biologically active compounds from plant material is largely
dependent on the type of solvent used in the extraction procedure (Hirun et al, 2012). The
solubility of polyphenols depends mainly on the hydroxyl groups, the molecular size and
the length of the hydrocarbon chain. The results of the phytochemical analysis from the
different extract-extractant combination are shown below:
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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Table 4. Physiologically active constituents present in the different plant extracts using
different extracting solvents
TESTS
RESULTS CONSTITUENTS
P
P
P P p g g g E E G
v
w h v w v w h v w v
1. Detection of Alkaloids
1.1Mayer’s Test
-
-
+ + + - - - + + - Alkaloids
1.2Dragendorff’s Test
-
-
- +
- + - + + - Alkaloids
2. Detection of glycosides
2.1Modified Borntrager’s -
-
- - + - - - - - - Anthraquinone
Test
glycoside
3. Detection of Phytosterol
3.1 Cupric Acetate Test
-
-
- - - + + + - - - Diterpenes
3.2 Salkowski’s Test
-
+ + - - + - + - - - Triterpenes
4.Detection of Phenolic
compounds
+ + + -
- - - - - - Phenolics
4.1 Ferric Chloride Test
-
-
- + - - - - + - + Tannins
4.2 Gelatin Test
5. Detection of Flavonoids
5.1 Alkaline Reagent Test + + + - + - - - + + + Flavonoids
5.2 Lead Acetate Test
+ + + -
+ + + + + + Flavonoids
Physiologically, investigations on the phytochemical analysis of the plant extracts
revealed the presence of bioactive and phenolic compounds such as alkaloids, diterpenes,
triterpenes, flavonoids, and phenolics, tannins and anthraquinones. These bioactive
constituents act as antimicrobial compounds. Quinones bind to adhesins, complex with cell
wall and inactivate enzymes. Flavonoids bind to adhesins, and complex with cell wall.
Polyphenols and tannins bind to adhesins, inhibits enzyme production, deprives substrate,
complexes with the cell wall, disrupts membrane and metal ion complexation. Terpenoids
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
and essential oils disrupt membrane. Alkaloids intercalate into cell wall and DNA and
inhibit release of autocoids and prostaglandins (Tiwari et al, 2011).
Results revealed that water can extract the following compounds: alkaloids
diterpenes, triterpenes and flavonoids while wine can extract the following compounds:
triterpenes, diterpenes and flavonoids. Vinegar, on the other hand, can extract diterpenes
and triterpenes, phenolics and flavonoids (Table 4). Since wine and vinegar contain a
significant amount of alcohol, it is assumed that bioactive compounds such as alkaloids
present in the solutions were changed into another derivative as a result of the organic
reactions formed by the alcohols present in the solvents, admonishing their greater
antibacterial activity in combination with the extracts as compared to water. Campbell and
Smith (2000) stated that when an alcohol reacts with a hemiacetyl group, the resulting
compound becomes glycoside. This was probably the phenomenon responsible for the
presence of anthraquinone glycoside in Pandanus sp. and wine combination. Plant extracts
from organic solvents have been found to give more consistent antimicrobial activity
compared to water extract because water soluble flavonoids (mostly anthocyanins) have
no antimicrobial significance and water soluble phenolics are only important as antioxidant
compounds (Das et al., 2010). Further, the capacity of the alcohol constituents of both
vinegar and wine to polarize the inner cellular membranes of the plants and extract more
active constituents is a possible explanation to why water and extracts alone cannot exhibit
greater antibacterial activity.
It is also interesting to note that although wine extracted more phytochemicals than
vinegar as in the case of P. betle whereby P. betle + wine extracted triterpenes, vinegar
was statistically proven to be the best extractant based from the inhibition zones measured.
Vinegar probably extracted a higher amount of phytochemical compounds than wine.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The study aimed to; 1. investigate the in-vitro activity of ten (10) microbial
antagonists against X. campestris pv. vitians, 2. determine the effect of each microbial
antagonists on the germination and seedling vigor of lettuce seedlings, 3. investigate the
in- vitro activity of five (5) aqueous plant extracts with three (3) different extracting
solvents at different concentrations against X. campestris pv. vitians and 4. identify the
phytochemicals using different extracting solvents.
Results of the in-vitro activity of ten bacterial antagonists against X. campestris pv.
vitians revealed the significant inhibitory effect of PCN-2011-004 on the growth of the
pathogen. This was comparable to the standard biofungicide, Virtuoso and Isolate 158.
Although lower in efficacy, PCN-2011-002 and PCN-2011-005 also effected a growth
suppression of X. campestris pv. vitians.
The highest germination was noted in Isolate 131. However, this was comparable
to all the microbial antagonists tested and the standard treatments. The lowest percentage
germination was obtained from the untreated seeds.
The highest mean height was noted in STR-2011-001 treated seeds. Nevertheless,
this did not significantly differ from Virtuoso, Isolate 131, Isolate 94, Isolate 158, PCN-
2011-003 and PCN-2011-005. On the other hand, Isolate 73, PCN-2011-002, PCN-2011-
004 and Isolate 31 gave significantly shorter seedlings than the above microbial antagonists
but differed significantly from the untreated seeds.
In the in-vitro activity of the extracts against the growth of X. campestris pv. vitians,
no significant interaction was observed among the treatments. Nevertheless, A. sativum
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
produced the highest mean followed by P. guajava, Euphorbia sp., P. betle and Pandanus
sp. Vinegar solvent gave the highest mean but was comparable to wine. However, this was
significantly different from water. The most effective concentration was 1:1 with a mean
at 15.13, comparable to 1:5 and 1:10 concentrations but differed significantly from
concentration 1:15.
Results of the phytochemical analysis revealed that water can extract the following
metabolites: alkaloids diterpenes, triterpenes and flavonoids; wine can extract the
following metabolites: triterpenes, diterpenes and flavonoids; and, vinegar can extract the
following metabolites: diterpenes and triterpenes, phenolics and flavonoids.
Conclusion
In-vitro tests showed that the microbial antagonists (MA’s) and organic-based
products tested in this preliminary experiment are potential biopesticides against X.
campestris pv. vitians. The promising microbial antagonists were Isolate 158 and PCN-
2011-004, for inhibiting the growth of the pathogen in-vitro; STR-2011-001 and Isolate
131 for stimulating the vigor of the lettuce seedlings in-vivo; and, Isolate 158 and PCN-
2011-005 for inhibiting the growth of the pathogen in-vitro and stimulating the growth of
the seedlings in-vivo, comparable to the standard Virtouso.
A. sativum combined with vinegar at 1:1 concentration is a potential organic-based
product against the said pathogen as its antimicrobial activity in-vitro in comparison to the
other isolates is more potent than that of the other treatments, regardless of which factor
they belong. Phytochemical analysis revealed that each organic solvent was able to extract
different kinds of phytochemicals from the cells of the leaves. However, in the sensitivity
tests, vinegar showed the highest inhibition zone at 1:1 concentration with no interference
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
from the pH. This indicates that the alcohol content of bignay vinegar can extract more
phytochemical compounds and retain their bonds to the organic phytochemicals when
unfavorable microchemical reactions occur.
Recommendations
Based from the results, the following are recommended:
1. Purify and identify the antibiotics present in the bacterial isolates showing
inhibition zones;
2. Extend the observation period in the assessment of ZOI (zone of inhibition) to
know the most effective application and devise a method or protocol of determining ZOI
for a period of five to seven days without necessarily affecting the efficacy of BCA’s;
3. Conduct compatibility tests between effective microbial agents and plant
extracts;
4. Devise a more rapid and easy extraction methods of extracting phytochemicals
from the effective plant materials;
5. Conduct greenhouse and field experiments to validate the efficacy of the
potential microbial antagonists and organic-based products against X. campestris pv.
vitians along with other lettuce diseases.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
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Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
MATIW, AGATE VICTRICIA P. MARCH 2013. Microbial Antagonists (MA’s)
and Organic-based Products as Potential Biopesticides against Black Leaf Spot
(Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa). Benguet State
University, La Trinidad, Benguet.
Adviser: Luciana M. Villanueva, Ph.D.
ABSTRACT
The study aimed to; 1. investigate the in-vitro activity of ten (10) microbial
antagonists against X. campestris pv. vitians, 2. determine the effect of each microbial
antagonists on the germination and seedling vigor of lettuce seedlings, 3. investigate the
in-vitro activity of five (5) aqueous plant extracts with three (3) different extracting
solvents at different concentrations against X. campestris pv. vitians, and 4. identify the
phytochemicals present in the extracts using different extracting solvents.
Results of the in-vitro activity of ten bacterial antagonists against X. campestris pv.
vitians revealed the significant inhibitory effect of PCN-2011-004 on the growth of the
pathogen. This was comparable to the standard biofungicide, Virtuoso and Isolate 158.
Although lower in efficacy, PCN-2011-002 and PCN-2011-005 also effected a growth
suppression of X. campestris pv. vitians.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
The highest germination was noted in Isolate 131. However, this was comparable
to all the microbial antagonists tested and the standard treatments. The lowest percentage
germination was obtained from the untreated seeds.
The highest mean height was noted in STR-2011-001 treated seeds. Nevertheless,
this did not significantly differ from Virtuoso, Isolate 131, Isolate 94, Isolate 158, PCN-
2011-003 and PCN-2011-005. On the other hand, Isolate 73, PCN-2011-002, PCN-2011-
004 and Isolate 31 gave significantly shorter seedlings than the above microbial antagonists
but differed significantly from sterile distilled water treated seeds.
The main effect of plant extracts on the growth of X. campestris pv. vitians was not
significant. Significant differences were however observed on the different extractants.
Vinegar was the most effective but it did not significantly differ from wine. The least
effective was water. On the other hand, the highest concentration (1:1) showed
significantly the highest inhibition zone. This was comparable to 1:5 and 1:10
concentrations but was significantly different from 1:15. No significant interaction was
noted among the factors evaluated.
The phytochemical analysis revealed that alkaloids, diterpenes, triterpenes and
flavonoids can be extracted by water. Triterpenes, diterpenes and flavonoids can be
extracted by wine. On the other hand, diterpenes, triterpenes, phenolics and flavonoids can
be extracted by vinegar.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
RESULTS AND DISCUSSION
Sensitivity Test of X. campestris pv. vitians to different microbial antagonists (MA’s)
Among the microbial antagonists tested, PCN-2011-004 gave the highest inhibition
zone followed by Isolate 158 with inhibition means of 4 and 3.33 mm, respectively (Table
1). The two microbial agents were comparable to Virtuoso, the standard treatment, with a
mean of 4.67 mm. Meanwhile, PCN-2011-005 with inhibition mean of 3.03 mm was
similar to PCN-2011-004, Isolate 158 and PCN-2011-002. No significant differences were
observed among the following treatments: sterile distilled water, Isolate 73, STR-2011-
001, Isolate 94, Isolate 31, Isolate 131, PCN-2011-003 and cuprous oxide as they did not
actively inhibit the growth of the pathogen. The two isolates comparable to Virtuoso can
be manipulated to either equal the antibiotic potency of Virtuoso or to overcome the latter’s
diffusion rate, in-vitro.
Antibiosis, the antagonism resulting from the production by one microorganism
Table 1. Efficacy of the different microbial antagonists, Virtuoso and cuprous oxide on the
growth of X. campestris pv. vitians
Treatment
Actual Mean
Transformed Mean
Sterile Distilled water
0.00d
0.71d
PCN-2011-004(Bacillus sp.)
4.00ab
4.00ab
PCN-2011-005 (Bacillus sp.)
2.33c
2.33c
Isolate 73 (B. pumilus)
0.00d
0.71d
PCN-2011-002 (Bacillus sp.)
3.03bc
3.03bc
STR-2011-001 (Bacillus sp.)
0.00d
0.71d
Isolate 94 (Flavobacterium sp.) 0.00d
0.71d
Isolate 31 (Bacillus sp.)
0.00d
0.71d
Isolate 131 (B. pumilus)
0.00d
0.71d
Isolate 158 (Pseudomonas sp.)
3.33abc
3.33abc
PCN-2011-003 (Bacillus sp.)
0.00d
0.71d
Virtuoso (B. subtilis)
4.67a
4.33a
Cuprous oxide
0.00d
0.71d
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
of secondary metabolites toxic to other microorganisms, is a form of microbial antagonism
wherein direct interaction between two microorganisms shares the same ecological niche
(Alabouvette et al., 2006). This derived mechanism accounts for the formation of clear
zones between the pathogen and the BCA’s. Further, Alabouvette et al stated that antibiosis
is a very common phenomenon responsible for the activity of many BCA’s such as
fluorescent Pseudomonas and Bacillus spp.
Fluorescent pseudomonads owe their fluorescence to an extracellular diffusible
pigment called pyoverdin (Pvd) or pseudobactin. This pigment has high affinity for Fe3+
ions and is a siderophore (iron-carrier) of the producer strain (Meyer and Abdallah, 1978).
In iron depleted media in vitro, Pvd-producing Pseudomonas spp. inhibited the growth of
bacteria with less potent siderophores (Kloepper et al., 1980), explaining the small halo
produced around the susceptible pathogen. Under certain conditions therefore, Pvd
functions as a diffusible bacteriostatic antibiotic.
The mechanism for growth suppression employed by Bacillus spp. against X.
campestris pv. vitians as observed was the production of antibiotics, although the type of
antibiotics produced in this case is not known. In fungal diseases, B. subtilis produced
mycosubtilin against Pythium aphanidermatum (Leclere et al., 2005) while B.
amyloliquefaciens produced bacillomycin and fengycin against Fusarium oxysporum
(Koumoutsi et al., 2004). Purification and identification therefore of the toxic metabolites
produced by the Bacillus species are necessary to understand fully the mechanism of
control expressed in vitro to be able to modify its structure and improve its durability as
this antibiotic expression can be lost over time.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
Germination rate, seedling height and vigor index as affected by the different microbial
antagonists
Isolate 131 gave the highest germination rate at 97.22% with significant differences
to Isolate 158, PCN-2011-003, PCN-2011-005, and PCN-2011-002 at 95.83%, 95.83%,
95.55%, and 91.67% respectively (Table 2). However, the latter isolates were comparable
to Isolate 31, STR-2011-001, Isolate 94, Isolate 73, Virtuoso, Cuprous oxide and PCN-
2011-004 at 90.27%, 90%, 88.87%, 86.11%, 83.34%, 81.94% and 81.94%. Further, the
latter isolates are not significantly different to the control.
Germination test on lettuce variety Tyrol revealed that Virtuoso stimulated
germination at 90% while cuprous oxide at 89.25% (Calderon, 2011). Results of which
were higher compared to what was obtained in this experiment. Since variables can be
manipulated, it can be inferred that the percentage germination expressed by the standard
treatments operate on numerical variables as 80-90% germination capacity.
Table 2. Influence of microbial antagonists on the germination and growth of lettuce
seedlings
Seedling
Seedling
Seedling
TREATMENTS
Germination
Height
Vigor Index
Percentage
(mm)
(%)
Sterile Distilled water
75.00b
67.07d
5033.40d
PCN-2011-004(Bacillus sp.)
81.94ab
82.73bcd
6809.38bcd
PCN-2011-005 (Bacillus sp.)
95.55a
90.80abc
8670.02ab
Isolate 73 (B. pumilus)
86.11ab
83.13bcd
7178.80abcd
PCN-2011-002 (Bacillus sp.)
91.67a
83.13bcd
7630.28abc
STR-2011-001 (Bacillus sp.)
90.00ab
102.40a
9219.50a
Isolate 94 (Flavobacterium sp.)
88.89ab
88.33abc
7856.47abc
Isolate 31 (Bacillus sp.)
90.28ab
82.53bcd
7449.39ab
Isolate 131 (B. pumilus)
97.22a
92.33ab
8929.56ab
Isolate 158 (Pseudomonas sp.)
95.83a
85.80abcd
8248.61ab
PCN-2011-003 (Bacillus sp.)
95.83a
85.80abcd
7838.89abc
Virtuoso (B. subtilis)
83.34ab
86.33abc
7166.49abcd
Cuprous oxide
81.94ab
72.87cd
5960.53cd
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
The ability of cuprous oxide to induce germination rate could be attributed to the
presence of growth promoting hormones such as IAA, auxins, cytokinins, and gibberellins
added to its over-all ingredient. Calderon (2011) citing an article from the agribusiness
week stated that copper is an essential microelement needed by plants to promote seed
production and formation. Maneb, for example, induces germination of seeds due to the
presence of Zn as one of its ingredients. BCA’s also produce Indole Acteic Acid (IAA),
auxins, cytokinins and gibberellins to promote growth. When Ahmad, et al (2006) screened
free-living rhizospheric bacteria for their multiple plant growth promoting activities,
Pseudomonas and Bacillus produced IAA, along with Azotobacter isolates.
In the determination of the effect of the biocontrol agents on the seedling height of
lettuce, STR-2011-001 promoted seedling growth at 102.40 mm, being the highest among
the bacterial agents, followed by Isolate 131, PCN-2011-005, Isolate 94, Virtuoso, Isolate
158, PCN-2011-003, Isolate 73, PCN-2011-004, Isolate 31, cuprous oxide and distilled
water at 92.33, 90.80, 88.33, 86.33, 85.80, 85.79, 83.13, 83.13, 82.73, 82.53, 72.87 and
67.07 mm, respectively.
Statistically, STR-2011-001 was comparable to Isolate 131, PCN-2011-005, Isolate
94, Virtuoso, Isolate 158, and PCN-2011-003 and significantly different to PCN-2011-002,
Isolate 73, PCN-2011-004, Isolate 31, cuprous oxide, and the control. These reactions
indicate that PGPR capacity of microbial agents does not necessitate that germination
stimulation and growth promotions are closely linked characteristics. One biocontrol agent
may stimulate germination; one may promote growth, while one may have both. Isolate
131 is an example.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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In the seedling vigor index tests, STR-2011-001 gave the highest index at 9219.50,
however it was comparable to Isolate 131, PCN-2011-005, Isolate 158, Isolate 94, PCN-
2011-003, PCN-2011-002, Isolate 31, Isolate 73, and Virtuoso at 8929.56, 8670.02,
8248.61, 7856.47, 7838.89, 7630.28, 7449.39, 7178.80, 7166.49 and differed significantly
from PCN-2011-004, cuprous oxide and the control with respective indexes of 6809.38,
5960.53 and 5033.40.
Note that Pseudomonas sp. (Isolate 158) gave an index of 8248.60. In the sensitivity
screening, its antibiotic behavior was linked to its production of siderophores. Schroth et
al (1982) postulated that the resulting siderophore hypothesis exert their plant growth
promoting activity by depriving pathogens of iron. An example cited by Haas and Defago
(2005) was the suppression of Fusarium wilt and take-all, by P. putida strain B10 but this
suppression was lost when the soil was amended with Iron; a move which repressed
siderophore production in this strain.
To follow, PGPR can also protect a crop against phyto diseases. Harman et al
(2004) stated that some biocontrol PGPR elicit a phenomenon known as Induced Systemic
Resistance (ISR) in the host plant. ISR is an indirect phenomenon expressed by any plant
in reaction to chemical molecules from natural or synthetic origins (Alabouvette et al.,
2005) which were assumed to be produced by the Bacillus and Pseudomonas strains
utilized in this study. For example, in Pseudomonas strains, a combination of pyocyanin
and pyochelin seems to be most effective for inducing resistance in tomato (Audenaert et
al., 2002). The plant growth stimulating volatile 2,3-butanediol that is found in Bacillus
spp. can also initiate ISR (Ryu et al., 2004).
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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Calderon (2011) revealed that bacterial isolate 9 induced germination at 95.5%,
comparable to Virtuoso and Trichoderma with treatment means of 91.5% and 90%
respectively. It also promoted seedling height with means of 48.60 mm although not
comparable to Trichoderma and Virtuoso with treatment means of 61.56 mm and 54.31
mm respectively. Interestingly, bacterial isolate 9 reduced black leaf spot severity with a
rating of 1.92, in comparison to the control (3.08) following Virtuoso at 1.83 rating. This
could illustrate the effect of PGPR on ISR of lettuce against X. campestris pv. vitians.
However, it should be noted that ISR and other modes of action are not mutually exclusive;
ISR might only exert a complementary effect to microbial antagonism (Alabouvette et al.,
2005).
Collectively, the results show two promising biological control agents with the
capacity to suppress growth of X. campestris pv. vitians in-vitro, stimulate germination and
promote seedling growth. These are PCN-2011-005 and Isolate158 as shown by table 3 in
comparison to the other standard treatments.
Statistically, Virtuoso produced the highest mean of inhibition at 4.33 and was
significantly different from Isolate 158, PCN-2011-005, cuprous oxide and the control.
Isolate 158 and PCN-2011-005 were however not significantly different from each other.
Table 3. Efficacy of promising microbial antagonists on the growth of X. campestris
pv. vitians and on the germination, seedling height and seedling vigor of lettuce
TREATMENTS
Inhibition %
Seedling
Seedling
Zone
germination height
vigor
(mm)
(mm)
index
T0 – SDW
0.00c
75.00b
67.07c
5033.40c
T2 – PCN-2011-005 (Bacillus sp.) 2.33b
95.55a
90.80a
8670.02a
T3 – Isolate 158 (Pseudomonas
3.33b
95.83a
85.80ab
8248.61ab
sp.)
T4– Virtuoso (B. subtilis)
4.67a
83.34ab
86.33ab
7166.49abc
T5 – Cuprous oxide
0.00c
81.94ab
72.87bc
5960.53bc
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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In terms of percentage germination, Isolate 158 was the highest at 95.83. This was
however not significantly different from PCN-2011-005. Both treatments were however
comparable to Virtuoso and cuprous oxide. In terms of seedling height, PCN-2011-005
produced the highest mean at 90.80, comparable to Isolate 158 and Virtuoso but
significantly different from distilled water and cuprous oxide. Cuprous oxide was however
comparable to Isolate 158 and Virtuoso. In terms of seedling vigor, PCN-2011-005
produced the highest mean at 8670.02, comparable to Isolate 158 and Virtuoso but
significantly different from distilled water and cuprous oxide. Cuprous oxide was however
comparable to Isolate 158 and Virtuoso.
Note however, that many effective biocontrol PGPR elicit ISR without necessarily
producing antibiotics (Zhender et al., 2001; Ongena et al., 2004; and Ton et al., 2002).
This could be true to both STR 1 and Isolate 131.
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MATIW, AGATE VICTRICIA P. MARCH 2013
a
b
c
d
e
f
g
h
i
j
k
l
m
Fig 1. Effect of microbial antagonists on the height of lettuce seedlings: a. Sterile
Distilled Water; b. PCN-2011-004; c. PCN-2011-005; d. Isolate 73; e. PCN-2011-
002; f. STR-2011-001; g. Isolate 94; h. Isolate 31; i. Isolate 131; j. Isolate 158; k.
PCN-2011-003; l. Virtuoso; m. cuprous oxide.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
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Sensitivity screening of X. campestris pv. vitians to different organic-based products at
different concentrations
Extract
a
a
12
a
10
a
a
8
6
Extract
4
2
0
Piper betle
Pandanus
Allium
Euphorbia
Psidium
sp.
sativum
sp.
guajava
Fig 2. Efficacy of extracts against X. campestris pv. vitians
Statistically, no significant difference was obtained from the means of the data
analyzed, but numerically, A. sativum was the best extract at 10.21, followed by P. guajava,
Euphorbia sp., P. betle, and Pandanus sp., at 9.22, 7.44, 6.56, and 5.52 respectively. The
greater antimicrobial activity of A. sativum could be attributed to the compound allicin, one
of the active principles of freshly crushed garlic homogenates. Allicin has a variety of broad
spectrum antimicrobial activities. In its pure form, it was found to inhibit Gram negative
bacteria causing human diseases (Ankri and Mirelman, 1999). Lirio et al (1999) reported
that garlic was able to inhibit the growth of X. campetsris pv. campestris, in-vitro. The
main antimicrobial effect of allicin is due to its chemical reaction with thiol groups of
various enzymes, e.g. alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase
which can affect essential metabolism of cysteine proteinase activity involved in the
virulence of some bacteria (Ankri and Mirelman, 1999). Its effect on X. campestris pv.
vitians is one concern raised during this study; although, Cavallito and Bailey (1945)
reported that inhibition of thiol-containing enzymes in the microorganism by the rapid
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
reaction of the thiosulfanates with thiol groups was assumed to be the main mechanism
involved in the antibiotic effect. It is reasonable to conclude, therefore, that the wide
spectrum antimicrobial effects of allicin are due to the multiple inhibitory effects they may
have on various thiol-dependent enzymatic systems (Ankri and Mirelman, 1999).
Berdy et al 1982 and Caceres et al 2005 attributed the antibiotic effect of P. guajava
to 2 compounds, guajaverin and psidiolic acid against bacteria. Agarwal et al (2012)
postulated that the antibacterial activity of leaves of P. betle against gram positive and gram
negative bacteria is due to the presence of metabolic toxins or broad spectrum antimicrobial
compounds. Euphorbia sp. and Pandanus sp. also have antibiotic compounds responsible
for the inhibition zones formed around the agar wells against X. campestris pv. vitians.
Generally, plants elicit a certain chemical as a form of defense mechanism against an
invasion by a foreign material.
Extractant
a
20
15
ab
Extractant
10
b
5
0
Water
Wine
Vinegar
Fig 3. Efficacy of water, wine and vinegar as organic solvents to extract phytochemical
compounds from the plant materials
Vinegar showed the highest mean at 15.07, followed by wine and water with means
of 6.57 and 1.73, respectively (Fig. 3). Statistically, vinegar was the most effective but this
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
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was comparable to wine and differed significantly with water. Vinegar contains acetic acid,
a form of ethanol responsible for the alcohol content of the vinegar. Wines in particular
may contain either ethanol or methanol. Alcohols are good solvents for the extraction of
phytochemicals situated within the plant cells as they are more efficient in cell wall
degradation causing polyphenols to be released from cells. The decrease in activity of
aqueous extract (water) can be ascribed to the enzyme polyphenol oxidase, which degrade
polyphenols in water extracts whereas in methanol and ethanol (alcohols) they are inactive
(Lapornik et al., 2005). Reasons as to why wine is lower than vinegar, although they have
alcohols, will be discussed further in the phytochemical analysis.
Fig 4 illustrates the effect of the concentration on the efficacy of the extracts.
Concentration
a
20
15
ab
10
Concentration
b
ab
5
0
1:01
1:05
1:10
1:15
1:1
1:5
Fig 4. Effect of concentrations on the efficacy of plant extracts against X. campestris pv.
vitians
Apparently, concentration 1:1 was the most effective but comparable to 1:5, 1: 10
and 1:15 with respective means of 15.14, 6.93, 4.80 and 4.29. The inference therefore is
that, as the dilution of solvents to the plant materials is increased, the efficacy of the
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
concentrations against X. campestris pv. vitians decreases. Vis-à-vis, as the dilution
decreases, the efficacy of the concentrations increases.
On the other hand, no significant interaction was observed among the three factors,
namely: extract, extractant and concentration.
A
1:1
1:5
1:10
1:15
B
1:1
1:5
1:10
1:15
C
1:1
1:5
1:10
1:15
Fig 5. Effect of different extracting solvents and concentrations on the efficacy of
P. betle against X. campestris pv. vitians: A. Water, B. Wine, C. Vinegar
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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A
1:1
1:5
1:10
1:15
B
1:1
1:5
1:10
1:15
C
1:1
1:5
1:10
1:15
Fig 6. Effect of different extracting solvents and concentrations on the efficacy of
Pandanus sp. against X. campestris pv. vitians; A. Water, B. Wine. C. Vinegar
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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A
1:1
1:5
1:10
1:15
A
1:1
1:5
1:10
1:15
A
1:1
1:5
1:10
1:5
Fig 7. Effect of different extracting solvents and concentrations on the efficacy of
l
A. sativum against X. campestris pv. vitians; A. Water, B. Wine, C. Vinegar
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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A
1:1
1:5
1:10
1:15
B
1:1
1:5
1:10
1:15
C
1:1
1:5
1:10
1:15
Fig 8. Effect of different extracting solvents and concentrations on the efficacy of
l
Euphorbia sp. against X. campestris pv. vitians; A. Water, B. Wine, C. Vinegar
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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A
1:1
1:5
1:10
1:15
A
1:1
1:5
1:10
1:15
A
1:1
1:5
1:10
1:15
Fig 9. Effect of different extracting solvents and concentrations on the efficacy of
P. guajava against X. campestris pv. vitians; A. Water, B. Wine, C. Vinegar
l
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
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Phytochemical analysis of the different plant extract - extractant combination
Extensive research has been performed worldwide and important evidences were
collected to show the immense potential of plants used in various traditional therapeutic
systems (Caunii et al., 2005). Many scientists nowadays are exploiting this complex line
of systems to answer the problems on resistance exhibited by pathogens to the existing
antibiotics. The exploitation of the botanical world begins with the understanding of the
phytochemicals present in the plant with specific concern to the bioactive compounds in
the plant materials tested against X. campestris pv. vitians.
The phenolic compounds present in extracts of plants are always a mixture of
different classes of phenols selectively soluble in the solvents (Caunii et al., 2005).
Successful determination of biologically active compounds from plant material is largely
dependent on the type of solvent used in the extraction procedure (Hirun et al, 2012). The
solubility of polyphenols depends mainly on the hydroxyl groups, the molecular size and
the length of the hydrocarbon chain. The results of the phytochemical analysis from the
different extract-extractant combination are shown below:
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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Table 4. Physiologically active constituents present in the different plant extracts using
different extracting solvents
TESTS
RESULTS CONSTITUENTS
P
P
P P p g g g E E G
v
w h v w v w h v w v
1. Detection of Alkaloids
1.1Mayer’s Test
-
-
+ + + - - - + + - Alkaloids
1.2Dragendorff’s Test
-
-
- +
- + - + + - Alkaloids
2. Detection of glycosides
2.1Modified Borntrager’s -
-
- - + - - - - - - Anthraquinone
Test
glycoside
3. Detection of Phytosterol
3.1 Cupric Acetate Test
-
-
- - - + + + - - - Diterpenes
3.2 Salkowski’s Test
-
+ + - - + - + - - - Triterpenes
4.Detection of Phenolic
compounds
+ + + -
- - - - - - Phenolics
4.1 Ferric Chloride Test
-
-
- + - - - - + - + Tannins
4.2 Gelatin Test
5. Detection of Flavonoids
5.1 Alkaline Reagent Test + + + - + - - - + + + Flavonoids
5.2 Lead Acetate Test
+ + + -
+ + + + + + Flavonoids
Physiologically, investigations on the phytochemical analysis of the plant extracts
revealed the presence of bioactive and phenolic compounds such as alkaloids, diterpenes,
triterpenes, flavonoids, and phenolics, tannins and anthraquinones. These bioactive
constituents act as antimicrobial compounds. Quinones bind to adhesins, complex with cell
wall and inactivate enzymes. Flavonoids bind to adhesins, and complex with cell wall.
Polyphenols and tannins bind to adhesins, inhibits enzyme production, deprives substrate,
complexes with the cell wall, disrupts membrane and metal ion complexation. Terpenoids
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
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and essential oils disrupt membrane. Alkaloids intercalate into cell wall and DNA and
inhibit release of autocoids and prostaglandins (Tiwari et al, 2011).
Results revealed that water can extract the following compounds: alkaloids
diterpenes, triterpenes and flavonoids while wine can extract the following compounds:
triterpenes, diterpenes and flavonoids. Vinegar, on the other hand, can extract diterpenes
and triterpenes, phenolics and flavonoids (Table 4). Since wine and vinegar contain a
significant amount of alcohol, it is assumed that bioactive compounds such as alkaloids
present in the solutions were changed into another derivative as a result of the organic
reactions formed by the alcohols present in the solvents, admonishing their greater
antibacterial activity in combination with the extracts as compared to water. Campbell and
Smith (2000) stated that when an alcohol reacts with a hemiacetyl group, the resulting
compound becomes glycoside. This was probably the phenomenon responsible for the
presence of anthraquinone glycoside in Pandanus sp. and wine combination. Plant extracts
from organic solvents have been found to give more consistent antimicrobial activity
compared to water extract because water soluble flavonoids (mostly anthocyanins) have
no antimicrobial significance and water soluble phenolics are only important as antioxidant
compounds (Das et al., 2010). Further, the capacity of the alcohol constituents of both
vinegar and wine to polarize the inner cellular membranes of the plants and extract more
active constituents is a possible explanation to why water and extracts alone cannot exhibit
greater antibacterial activity.
It is also interesting to note that although wine extracted more phytochemicals than
vinegar as in the case of P. betle whereby P. betle + wine extracted triterpenes, vinegar
was statistically proven to be the best extractant based from the inhibition zones measured.
Vinegar probably extracted a higher amount of phytochemical compounds than wine.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
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SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The study aimed to; 1. investigate the in-vitro activity of ten (10) microbial
antagonists against X. campestris pv. vitians, 2. determine the effect of each microbial
antagonists on the germination and seedling vigor of lettuce seedlings, 3. investigate the
in- vitro activity of five (5) aqueous plant extracts with three (3) different extracting
solvents at different concentrations against X. campestris pv. vitians and 4. identify the
phytochemicals using different extracting solvents.
Results of the in-vitro activity of ten bacterial antagonists against X. campestris pv.
vitians revealed the significant inhibitory effect of PCN-2011-004 on the growth of the
pathogen. This was comparable to the standard biofungicide, Virtuoso and Isolate 158.
Although lower in efficacy, PCN-2011-002 and PCN-2011-005 also effected a growth
suppression of X. campestris pv. vitians.
The highest germination was noted in Isolate 131. However, this was comparable
to all the microbial antagonists tested and the standard treatments. The lowest percentage
germination was obtained from the untreated seeds.
The highest mean height was noted in STR-2011-001 treated seeds. Nevertheless,
this did not significantly differ from Virtuoso, Isolate 131, Isolate 94, Isolate 158, PCN-
2011-003 and PCN-2011-005. On the other hand, Isolate 73, PCN-2011-002, PCN-2011-
004 and Isolate 31 gave significantly shorter seedlings than the above microbial antagonists
but differed significantly from the untreated seeds.
In the in-vitro activity of the extracts against the growth of X. campestris pv. vitians,
no significant interaction was observed among the treatments. Nevertheless, A. sativum
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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MATIW, AGATE VICTRICIA P. MARCH 2013
produced the highest mean followed by P. guajava, Euphorbia sp., P. betle and Pandanus
sp. Vinegar solvent gave the highest mean but was comparable to wine. However, this was
significantly different from water. The most effective concentration was 1:1 with a mean
at 15.13, comparable to 1:5 and 1:10 concentrations but differed significantly from
concentration 1:15.
Results of the phytochemical analysis revealed that water can extract the following
metabolites: alkaloids diterpenes, triterpenes and flavonoids; wine can extract the
following metabolites: triterpenes, diterpenes and flavonoids; and, vinegar can extract the
following metabolites: diterpenes and triterpenes, phenolics and flavonoids.
Conclusion
In-vitro tests showed that the microbial antagonists (MA’s) and organic-based
products tested in this preliminary experiment are potential biopesticides against X.
campestris pv. vitians. The promising microbial antagonists were Isolate 158 and PCN-
2011-004, for inhibiting the growth of the pathogen in-vitro; STR-2011-001 and Isolate
131 for stimulating the vigor of the lettuce seedlings in-vivo; and, Isolate 158 and PCN-
2011-005 for inhibiting the growth of the pathogen in-vitro and stimulating the growth of
the seedlings in-vivo, comparable to the standard Virtouso.
A. sativum combined with vinegar at 1:1 concentration is a potential organic-based
product against the said pathogen as its antimicrobial activity in-vitro in comparison to the
other isolates is more potent than that of the other treatments, regardless of which factor
they belong. Phytochemical analysis revealed that each organic solvent was able to extract
different kinds of phytochemicals from the cells of the leaves. However, in the sensitivity
tests, vinegar showed the highest inhibition zone at 1:1 concentration with no interference
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
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from the pH. This indicates that the alcohol content of bignay vinegar can extract more
phytochemical compounds and retain their bonds to the organic phytochemicals when
unfavorable microchemical reactions occur.
Recommendations
Based from the results, the following are recommended:
1. Purify and identify the antibiotics present in the bacterial isolates showing
inhibition zones;
2. Extend the observation period in the assessment of ZOI (zone of inhibition) to
know the most effective application and devise a method or protocol of determining ZOI
for a period of five to seven days without necessarily affecting the efficacy of BCA’s;
3. Conduct compatibility tests between effective microbial agents and plant
extracts;
4. Devise a more rapid and easy extraction methods of extracting phytochemicals
from the effective plant materials;
5. Conduct greenhouse and field experiments to validate the efficacy of the
potential microbial antagonists and organic-based products against X. campestris pv.
vitians along with other lettuce diseases.
Microbial Antagonists (MA’s) and Organic-based Products as Potential Biopesticides
against Black Leaf Spot (Xanthomonas campestris pv. vitians) of Lettuce (Lactuca sativa)|
MATIW, AGATE VICTRICIA P. MARCH 2013
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