BIBLIOGRAPHY AGAPITO, EMERLOU P. APRIL...
BIBLIOGRAPHY
AGAPITO, EMERLOU P. APRIL 2012.Evaluation of Bacteria and Actinomycetes-like
Organisms against Club Root (PlasmodiophoraBrassicae) in Chinese Cabbage
(BrassicaPekinensis). Benguet State University, La Trinidad, Benguet.
Adviser: Asuncion L. Nagpala. PhD.
ABSTRACT
The study was conducted at the department of plant pathology green house, Benguet
State University, LaTrinidad, Benguet from April 2011to January 2012to evaluate the effect of
bacterial isolates and actinomycetes-like organismson the growth and development of Chinese
cabbage and their effects on the incidence and severity of club root.
Seedling experiment revealed thatsix isolates from the dilution of 104, six isolates from
the dilution of 105and three isolates from the dilution of 106enhanced seed germination rate of
chinese cabbage to 100%. Likewise, isolates 9 and 10sigificantly improved the height of
seedlings using dilutions 104,105and106.
On the other hand, pot experiment showed that isolates 3, 4, 5,6,1 and 9 significantly
increased the nitrogen content of the soil after harvest. In addition, isolates 10, 3 and 9 gave the
lowest percent club root infection of 26.66 %, 40 % and 46.67%. The same isolates gave the
lowest club root severity infection of 2.3 % and 2.67 %. In terms of vegetative fresh weight,
isolate 1 gave the highest.
From the results, some bacterial isolates and actinomycetes-like organism from sunflower
soil improved seed germination rate and plant height, enhanced nitrogen uptake and reduced club
root severity.
Evaluation of Bacteria and Actinomycetes‐like Organisms against Club Root (PlasmodiophoraBrassicae) in
Chinese Cabbage (BrassicaPekinensis) / Emerlou P. Agapito. 2012
TABLE OF CONTENTS
Page
Bibliograpy…………………………………………………………………….......... i
Abstract……………………………………………………………………………… i
Table of Contents……………………………………………………………………
ii
INTRODUCTION……………………………………………………………………. 1
REVIEW OF LITERATURE…………………………………………………………
4
The Nomenclature and Classification
of the Actinomycetes…………………………………………………………
4
Isolation, Identification, Cultivation
and Preservation……………………………………………………………...
5
Nitrogen Fixation……………………………………………………………..
7
Activity of Actinomycetes
upon Plant Pathogenic Fungi……………………………………………......
8
Causation of Plant Diseases………………………………………………….
8
Plant Growth Promotion Activity
ofSecondary Metabolites………………………………………………………
9
MATERIALS AND METHODS……………………………………………………..
10
Seedling Experiment…………………………………………………………
10
Pot
Experiment………………………………………………………………..
11
Data Gathered…………………………………………………………………
13
RESULTS AND DISCUSSION……………………………………………………… 15
Seedling Experiment…………………………………………………………………... 15
Percentage Seed Germination
asAffected by the Different isolates……………………………………….......
15
Percentage Seed Germination
asAffectedby the Different Dilution Rates……………………………………
15
Interaction Effect on the Seed
15
Germination…………………………………………………………………..
Seedlings Height as Affected
16
bythe Different Isolates………………………………………………………
Seedlings Height as Affected
17
by the Different Dilution Rates……………………………………………….
Interaction
Effect
17
on the Seedlings Height………………………………………………………
Seedlings Root Length as Affected
bythe Different Isolates………………………………………………………
19
Seedlings Root Length as Affected
by the Different Dilution Rates………………………………………………
19
Interaction
Effect on the Seedlings
Root Length…………………………………………………………………..
20
Pot experiment…………………………………………………………………………
21
Nitrogen Content of the Soil………………………………………………..
21
Final
Height……………………………………………………………………
22
Percentage Incidence of Club root…………………………………………….
22
Club Root Severity……………………………………………………………
23
Fresh Weights of Vegetative Parts……………………………………………..
24
Dry Weights of Vegetative Parts……………………………………………… 26
Fresh Roots Weight…………………………………………………………..
27
Roots Dry Weight……………………………………………………………
28
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary……………………………………………………………………… 29
Conclusion ……………………………………………………………………
30
Recommendations……………………………………………………………. 31
LITERATURE CITED………………………………………………………………..
31
APPENDICES……………………………………………………………………….. 33
1
INTRODUCTION
Non adverse effects on the environment of biocontrol strategies of pest
management are priorities of tomorrow’s world agriculture (Baniasadi, 2009). Biological
control is slow but can be long lasting, inexpensive, and harmless to living organisms and
the ecosystem. It neither eliminates the pathogen nor the disease, but brings them into
natural balance. Intensive research on plant growth promoting bacteria (PGPB) is
underway worldwide for developing biofertilizers and biocontrol agents (BCAs) as better
alternatives to chemicals (Ningthoujam, 2009). High input agriculture is increasingly
recognized as contributing to the degradation of environment and health besides
demanding high costs due to its dependence on chemical inputs (Sulastri, 2005).
Biocontrol with beneficial bacteria is one promising alternative to fungicides.
Hydrolases such as chitinase contribute to degradation of fungal cel wal s. Chitin is the
second most abundant polysaccharide in nature and a major component of fungal walls,
insect exoskeletons and crustacean shells. Chitinase secreted by biological control agents
is likely to be effective against pathogenic fungi which cel wal s are mainly made up of
chitin (Ningthoujam, 2009).Actinomycetes are active biocontrol agents due to their
antagonistic properties against wide range of plant pathogenic fungi (Baniasadi, 2009).
This group of microorganisms is best known for their ability to produce bio-active
metabolites including antibiotics, plant growth factors, and other substances. Many of the
presently used antibiotics such as streptomycin, gentamicin, rifamycin and erythromycin
are the product of Actinomycetes (Jeffrey, 2008).
Evaluation of Bacteria and Actinomycetes-like Organisms against Club Root
(PlasmodiophoraBrassicae) in Chinese Cabbage (BrassicaPekinensis) / Emerlou P. Agapito. 2012
2
Among Actinomycetes, the Streptomycetes are the dominant. The
non‐streptomycetes are called rare Actinomycetes, comprising approximately 100 genera
(Sivakumar, 2008). Streptomycesand other Actionmycetes are major contributors to
biological buffering of soils and have roles in organic matter decomposition conducive to
crop production.
Actinomycetesare responsible for much of the digestion of resistant carbohydrates
such as chitin and cellulose bioremediation. The number and types of Actinomycetes
present in a particular soil would be greatly influenced by geographical location such as
soil temperature, soil type, soil pH, organic matter content, cultivation, aeration and
moisture content. Some are able to grow at elevated temperatures (>50°C) and are
essential to the composting process (Burge, 2008).Population ofActinomycetes is
relatively lower than other soil microbes and contains a predominance of Streptomyces
that are tolerant to acid conditions. Arid soils of alkaline pH tend to contain fewer
Streptomyces and more of the rare genera such as Actinoplanes and Streptosporangium.
However, alkaliphilicActionmycetes will provide a valuable resource for novel products
of industrial interest, including enzymes and antimicrobial agents. Among
Actinomycetes, the Streptomyces are especially prolific. A search of the recent literature
revealed that at least 4,607 patents have been issued on Actinomycete related product and
processes. Streptomycescovers around 80% of total antibiotic product, with other genera
trailing numerical y. Micromonospora is second with less than one-tenth as many as
Streptomyces(Arifuzzaman, 2010).
3
The result that will be generated in this study whereby a new bacterial isolates
that wil be found effective as an enhancer of nutrients in the soil will contribute for the
growth and development of plants. Moreover the isolates that wil be found effective as
biological agent against club root will be added to the few existing bio control agents
used to manage the disease.
This study aimed to:
1. determine the effects of bacteria and Actinomycetes-like organisms on the
growth and development of chinese cabbage, and
2. determine their effects on the incidence and development of club root on
Chinese cabbage.
This study was conducted at the department of plant pathology green house,
Col ege of Agriculture, Benguet State University, La Trinidad, Benguet from April 2011
to January 2012.
4
REVIEW OF LITERATURE
The Nomenclatureand Classification
of theActinomycetes
Family Streptomycetaceae.Actinomycetes with branched slender mycelium that is
rarely or not septate forming spores on aerial hyphae and not fragmenting intooidia.
There are two genera, Streptomyces and Micromonospora(Waksman and Henrici, 1943)
Genus Streptomyces.Streptomycetaceae forming spores in chains on aerialhyphae
cal ed sporosphores. Sporosphores are apparently endogenous in origin, formed by a
segregationof protoplasm within the hypha into a series of round, oval or cylindric
bodies.Chains of spores are often spirally coiled. Sporophores may be simple orbranched.
The type of species of this newly-named genus is Streptomyces albus (Rossi-Doria
emend Krainsky) comb. nov. This species was formerly known as
ActinomycesalbusKrainsky and first described as StreptothrixalbaRossi-Doria. This is
one of the most common and best known species of the group. It is colorless with white
aerial mycelium, forming ovoidal spores in coiled chains on lateral branches of the aerial
hyphae. It is proteolytic, liquefying gelatin and peptonizing milk with the production of
alkaline reaction in the latter. It does not produce any soluble pigment eitheron an organic
or synthetic medium, but does produce a characteristic earthy ormusty odor (Waksman
and Henrici, 1943).
5
Genus Micromonospora.The name MicromonosporaOrskov, apply to those forms
which producing single conidia on lateral branches. Tsiklinskyhad previously applied the
name Thermoactinomyces to species of thisgroup, whose identity is clear from
photomicrographs. But in her descriptionof the genus she also included thermophilic
species with catenulate spores, basingthe genus on temperature relations rather on
morphology (Waksman, 1959).
Isolation, Identification, Cultivation,
and Preservation
Most of the techniques used in the isolation and cultivation of bacteria and
fungi also apply to Actinomycetes. The isolation of these organisms from soils and other
natural substrates is brought about by first plating out such materials in proper dilutions
on suitable agar or gelatin media. The plates are incubated at favorable temperatures, for
2 to 7 days, and the colonies picked and transferred to sterile liquid or solid media for
further development. A colony of an Actinomycetediffers from a bacterial colony due to
the presence of a filamentous extension of the original cell or cel s, spores, and
degradation products. It is not an accumulation of cel s originating from one or more
similar cells. These are compact, often leathery, giving a conical appearance, and have a
drysurface. According to Titus and Pereira (2005) the leathery or powdery appearance of
Actinomycetes colonies is due to the production of conidia. These are often covered with
aerial mycelium. When grown in liquid culture, either in a stationary or in a submerged
condition, the majority of Actinomycetes, notably members of the genera Streptomyces
and Micromonospora, grow in the form of flakes or spherical compact masses, leaving
the medium clear. The mass of growth can easily be removed by filtration through
6
ordinary paper. Only when growth undergoes lysis do the cellsdisintegrate completely
and a certain degree of turbidity occurs (Waksman, 1959).
The methods of studying the Actinomycetes population of soil, water, compost,
and other materials include microscopic observations,plate culture studies,and selective
culture procedures. Primarily for characterization and identification purposes,a standard
media, comprising both synthetic and organic, are most essential. Synthetic, chiefly
inorganic, media have found extensive application in the study of the morphology,
physiology, and cultural characterization of these organisms. Organic media are used for
obtaining supplementary evidence of a cultural nature, especially for strains that do not
grow at al or grow only very quickly on the common inorganic media. A Media used
primarily for obtaining maximum growth, especially for the maximum production of
certain chemical substances, such as antibiotics, vitamins, or enzymes are usually
complex in composition, utilizing plant and animal materials directly or after preliminary
enzymatic or acid digestion. For maintaining cultures of Actinomycetes in such a manner
as to reduce, to a minimum, degeneration and variationof the culture a suitable media,
comprising both artificial and natural, such as sterile soil, and suitable conditions of
growth thus make possible for the preservation of type cultures for comparative purposes.
The great majority of Actinomycetes are aerobic and very few are anaerobic and many
are microaerophilic. To supply proper aeration, the organisms are grown on the surface of
solid media, or in shallow liquid layers, or in a thoroughly aerated submerged condition.
For anaerobic growth, special procedures are required. Temperatures of 25-30° C are
usual y used for incubation of the great majority of Steptomyces, Nocardias, and
7
Micromonosporas. Pathogenic organisms require 37° C, and thermopiles usually require
50-60° C (Waksman, 1959).
Nitrogen Fixation
Various reports have been made in the past of the ability of one or more
Actinomycetes to fix atmospheric nitrogen. Meyen first observed nodules on alder roots
whichwas confirmed by Woronin. Brunchorst named the microbe microbes inside the
nodulesFrankiasubtilis and Hiltner recognized the nodule inhabitant as an actinomycete,
gram-positive bacteria closely related to Streptomyces. Like Streptomyces, Frankiaforms
spores, but it also produces structures known as vesiclesthat sequester the oxygen-labile
enzyme nitrogenase. The vesicle cell walls are composed ofhopanoid lipids, making them
impervious to oxygen. Itappears phase-bright under phasecontrast microscopy. Several
different Frankiastrains were alsoisolated from actinorhizal plants, including Casuarina,
Elaeagnus,and Myrica. Other Actinomycetes were also being isolated from the nodules
of diverse actinorhizalplants, but not much attention was being paid to them. In the late
1980’s, several Actinomyceteshad been isolated from nodules of Casuarinatrees
(indigenous to Australia) growing in Mexico (Hirsch, 2009).
twonocardias, N. calcarca and N. cdlulans, isolated from grassland lime soils
were found to have the capacity to fix atmospheric nitrogen to the extent of 2.0 to 4.5 mg
of X/gm of glucose or other carbon source in the medium. The second culture was also
capable of decomposing cel ulose, the amount of nitrogen fixed being 5 to 12 mg of
X/gm of cellulose decomposed (Waksman, 1959).
8
Activity of Actinomycetes
upon Plant Pathogenic Fungi
An extensive literature has accumulated upon the antagonistic effects of
Actinomycetes upon fungi, especially upon plant pathogens. Winterpresented further
evidence concerning the ability of various Actinomycetes to attack Ophiobolusgraminus,
an important parasite that attacks wheat. Sanford and Cormack tested the effect of eight
cultures of Actinomycetes upon the disease-producing fungus Helminthosporiumsativum.
In comparison with a disease rating of 66 percent for the untreated pathogen, four
Actinomycetes suppressed the virulence of the pathogen to 33, 22, and 1 per cent,
respectively; two had no marked effect; and the other two appeared to increase the
virulence by 12 and 16 percent, respectively. Perrault demonstrated that the growth of
Colletotrichumsepedonicum in agar media was impeded by several microorganisms
isolated from potato tubers affected with ring rot. Four of these organisms were
Actinomycetes and were able to produce antibiotic substances that diffused readily
through the medium and prevented al growth of the pathogen. One culture produced a
lysis of the plant pathogen (Waksman, 1959). In 2005 plant pathology journal, 10 isolates
of Actinomycetes was reported to have an antagonistic reaction to a single isolate of
Alternariasolani through agar plate method (ShahidiBonjar, 2005).
Causation of Plant Diseases
In spite of the great importance of Actinomycetes in nature, especially in the
soil, the number of plants attacked by these, as compared to the number of plants attacked
by bacteria, fungi, and viruses, is rather limited. Two species of plant which is the Irish
potato and the sugar beet plants are known to be infected by Actinomycetes and causes
9
scab. In Hoffmann’s work, numerous infection experiments were carried out with twenty
Streptomycesspecies, using a number of scab-susceptible potato varieties in the
greenhouse and under field conditions. He found out thatS. scabies was the pathogen of
potato scab and the same was true with beet scab (Waksman, 1959).
Plant growth Promotion Activity
of Secondary Metabolites
Although Actinomycetes produce numerous kinds of secondary metabolites, their
plant bioactivity is known very little. In their recent studies,Igarashi et al. (2006) have
identified at least 10 chemical y different classes of secondary metabolites produced by
Streptomyces hygroscopicus. Of these compounds, pteridic acid A induced the
adventitious root formation of kidney bean hypocotyls and growth promotion of tobacco
BY-2 cells which suggest the possible involvement of secondary metabolites in plant
growth promotion.
10
MATERIALS AND METHOD
Sterilized forest soil obtained at the forest area of Long long, Puguis, la Trinidad,
Benguet was used in the entire experiment.The sterilization period using steam pressured
drum lasted for 8 hours.
Seedling Experiment
Sterilized soil was distributed in a seedling tray with 3 x 13 holes. Different
dilutions of 104, 105 and 106 of the different isolates were prepared and 3ml each was
inoculated in seedling trays.The control was inoculated with water only. The experiment
set up fol owed the Complete Randomized Factorial Design (CRD Factorial) and was
replicated four times with 34 sample plants per treatment per replicate. Chinese cabbage
seeds were sown two weeks after inoculation. The treatments are shown below.
Treatments:
Factor A - Isolates
T0 -no bio-control added
Factor B – Dilution Rates
T1 - bacterial isolate 1
T2 - bacterial isolate 2
T3- bacterial isolate 3
T4- bacterial isolate 4
T5 - bacterial isolate 5
T6 - bacterial isolate 6
T7 -bacterial isolate 7
T8-bacterial isolate 8
T9-bacterial isolate 9
T10-bacterial isolate 10
11
Pot Experiment
The dilution of the different isolates that showed good performance during the
seedling experiment was further evaluated in a pot experiment. An amount of six kg of
soil were filled in pots measuring 6x6x11 inches and were inoculated with 30 ml 105
(best dilution) of the different isolates. Two weeks after the introduction of bacterial
isolates, every pot was infested with 5mlPlasmodiophorabrassicaehaving
sporeconcentration of 1x106 per ml.Seedlings that were taken from the seedling
experiment were transplanted in the inoculated pots one week after infestation. The
experiment set up utilized the complete randomized design (CRD) with 3 replicates
having 5 sample plants per treatment per replicate. The treatments are described below
while figure 1 shows the isolates used:
Treatments:
T0 -no bio-control added
T1 - bacterial isolate 1
T2 - bacterial isolate 2
T3- bacterial isolate 3
T4- bacterial isolate 4
T5 - bacterial isolate 5
T6 - bacterial isolate 6
T7 -bacterial isolate 7
T8-bacterial isolate 8
T9-bacterial isolate 9
T10-bacterial isolate 10
12
Figure 1. Colonies of the bacterial isolates used in the seedling
and pot experiment
13
Data Gathered:
A. Seedling experiment
1. Percentage seed germination. Germinated seeds was counted two weeks
aftersowing. Percent germination was determined using the formula,
no.f germinated seds
no.f seds own x 10
2. Seedlingheight. Height (inches) of al the 34 samples seedlings per replicate were
measured after six weeks.
3. Seedling root length. Roots of three sample plants were uprooted randomly were
measured.
B. Pot experiment
1. Nitrogen analysis of the soil. Soil samples of each replicate of the different
treatment weighing 200 grams was col ected after which was mix to come up with a
composite soil sample. The percentage N content(%N) of the soil wasestimated by
determining the organic matter content (%OM) of the soil multiplied by 0.05(%N=
%OM x 0.05). Soil analysis was done before the inoculation of bacterial isolateand after
harvest.
2. Final Height. Plant height of five samples was measured in inches one day before
theharvest.
3. Club Root Incidence. Plants infected with of club root was counted at harvest and
percent club root incidence was determined using the formula:
no.f sample plants −no.f healthy plants
% Incidence =
toal no.f plants
x 100
14
4. Club root Severity. Assessment of club root severity was based on the rating scale
of Anderson et al. as stated below:
Rating
Description
1
Normal root
2
Minor lateral clubbing at 0.5 cm diameter
3
Minor lateral clubbing at 1.2 cm diameter
4
Moderate clubbing
5
Severe clubbing in the tap root
6
Root decaying due advance infestation with plant
death.
5. Fresh and dry weights of vegetative parts. Fresh weights in grams of the
vegetative part was recorded before drying, whilethe dry weight was taken seven weeks
after air drying.
6. Fresh and dry weights of below ground parts. Fresh weights in grams of the
below ground partswas recorded before drying, while the dry weight was taken seven
weeks after air drying.
15
RESULTS AND DISCUSSION
A. Seedling Experiment
Percentage Seed Germination
asAffectedby the Different Isolates
According to Lindgren (1992), the standard percentage seed germination in
chinese cabbage is 80 % under optimum temperature.Table 1showsthe result of
germination rate two weeks after sowing.
The highest germination rate of 100% was recorded under isolate 4.Isolate 1
provided the second highest germination rate of 98.95% fol owed by isolates 2, 7 and 9.
Such result indicates that these isolates can be used as soil inoculants to improve seed
germination for they gave a better rate of germination than the control (96.86 %)
Percentage Seed Germination
asAffectedby the Different Dilution Rates
The highest germination rate of 98.12% and 97.18 % was recorded from the
dilutions of 104and 105.This means that higher concentration of isolates work effectively
in improving the germination rate of Chinese cabbage.
Interaction Effect on the Seed
Germination
Interaction between the factors suggest that al isolateimproved the germination
rate of chinese cabbage using dilutions of 104and 105 when inoculated in the soil before
sowing the seeds.Isolates withthe dilution of 106hadthe least percentage germination.
16
Table 1. Percentage germination as affected by the different isolates and the
differentdilution ratesrecorded after two weeks
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
96.86%
Isolate 1
100%
100%
96.86%
98.95%
Isolate 2
100%
100%
93.75%
97.92%
Isolate 3
100%
90.66%
100%
96.89%
Isolate 4
100%
100%
100%
100%
Isolate 5
90.66%
100%
96.86%
95.84%
Isolate 6
96.86%
96.86%
93.73%
95.82%
Isolate 7
100%
100%
93.75%
97.92%
Isolate 8
96.86%
87.5%
96.86%
93.74%
Isolate 9
96.86%
96.86%
100%
97.91%
Isolate 10
100%
100%
87%
95.67%
MEAN
98.124%
97.188%
95.881%
SeedlingsHeight as Affected
bythe Different Isolates
In terms of seedling height, Table 2 revealed that soils inoculated with isolate10
gave the tal est seedlings with the average height of 13.82 inches. This wasfollowed by
sample plantsunder isolates 3,9 and 4surpassing the control which means that thesecan
significantly enhancethe seedling height of chinese cabbage. Isolates 7, 1, and 6 slightly
increase the height of chinese cabbage.
17
Seedlings Height as Affected
bythe Different Dilution Rates
statistical analysis for the effect of different dilution rates (Table 2) shows that the
best dilutions that that can enhance the seedling height were 105and 106.
Interaction Effect on the Seedlings Height
Isolates 10, 3, 9 and 4 significantly improved the growth of chinese cabbage
seedlings using any of the dilutions while. Seedlings under isolate 10 with the dilution of
105 had the highest measurement of height.
Table 2: Seedling height (inches) as affected by the different isolates and the different
dilution rates measured after six weeks
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
2.51
Isolate 1
2.99
4.01
3.79
3.60d
Isolate 2
2.86
3.23
3.55
3.21d
Isolate 3
3.98
4.26
4.25
4.16b
Isolate 4
4.98
3.73
3.32
4.01d
Isolate 5
2.71
3.28
3.53
3.17d
Isolate 6
3.11
4.01
3.66
3.59cd
Isolate 7
3.17
3.72
3.99
3.63d
Isolate 8
2.96
3.53
3.29
3.26d
Isolate 9
3.63
4.42
4.20
4.08bc
Isolate 10
4.15
5.01
4.66
4.61a
MEAN
3.454b
3.92a
3.824a
Means with the same letter are not significantly different at 5% level DMRT
18
Figures 2 to 12 show the different set-up inoculated with isolates at different
dilution rate.
Figure 2. Seedlings not inoculated with
Figure 3. Chinese cabbage seedlings inoculated
bacterial isolates (water)
with bacterial isolate 1
Figure 4. Chinese cabbage seedlings
Figure 5. Chinese cabbage seedlings inoculated
inoculated with bacterial isolate 2
with bacterial isolate 3
Figure 6. Chinese cabbage seedlings inoculated
Figure 7. Chinese cabbage seedlings inoculated
with bacterial isolate 4
with bacterial isolate 5
Figure 8. Chinese cabbage seedlings
Figure 9. Chinese cabbage seedlings inoculated
inoculated with bacterial isolate 6
with bacterial isolate 7
19
Figure 10. Chinese cabbage seedlings
inoculated with bacterial isolate 8 Figure 11. Chinese cabbage seedlings
inoculated with bacterial isolate 9
Figure 12. Chinese cabbage seedlings inoculated
with bacterial isolate 10
SeedlingsRoot Length as Affected
Bythe Different Isolates
Table 3shows that al isolates enhanced the root length of chinese cabbage
seedling by surpassing the control. Isolate 6provided the highest root length mean of
11.37fol owed by isolate 5.
SeedlingsRoot Length as Affected
by the Different Dilution Rates
Result obtained for the effect of different dilutions on the root length of chinese
cabbage is almost the same which suggest that al dilution rates has no significant effect
on the root development of chinese cabbage.
20
Interaction Effect on the Seedlings
Root Length
The interaction between two factors suggests that isolate 6 with the dilution of
105wil improve the root development of roots in terms of length giving the highest mean
of 11.64. Main while dilution of 105used for isolate 7 gave the lowest mean of 8.25.
Table 3.Root length (inches) as affected by the different isolates and the different dilution
rates measured after six weeks
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
07.62
Isolate 1
09.91
10.23
08.43
09.52
Isolate 2
10.15
09.73
10.89
10.26
Isolate 3
08.28
09.57
09.80
09.22
Isolate 4
10.78
11.06
10.98
10.94
Isolate 5
11.04
10.44
11.59
11.02
Isolate 6
10.68
11.64
11.80
11.37
Isolate 7
09.71
08.25
10.69
09.55
Isolate 8
10.86
11.48
10.23
10.86
Isolate 9
10.78
11.38
10.46
10.87
Isolate 10
11.04
09.54
11.37
10.65
MEAN
10.32
10.33
10.62
21
B. Pot Experiment
Based on the result of seedling experiment, al isolates has a good effect on the
growth and development of chinese cabbage seedling. Using different rate of dilutions,
isolates with the dilution of 105 showed the most significantresult. This served as the
standard dilution rate for al isolate that was evaluated in the pot experiment.
Nitrogen Content of the Soil
Based on the result of % N content analysis as shown in Table 4, nitrogen content
al soil samples that were inoculated with bacterial isolatehad increased. Initial soils
withanitrogen content of 0.23 %,had significantlyincreased to0.34 %with the inoculation
of isolate 3. On the other hand, isolate 8 slightly increase the amount of nitrogen in the
soil.Under control, no significant increase can be observed.
Table 4.Initial and final percentage nitrogen content of soil samples, taken before the
inoculation of bacterial isolatesand after harvest
TREATMENT
REPLICATES
TOTAL
MEAN
S
R1
R2
R3
Initial soil
0.23
0.25
0.22
0.70
0.23c
Control
0.25
0.24
0.23
0.72
0.24bc
Isolate 1
0.30
0.32
0.31
0.93
0.31a
Isolate 2
0.30
0.31
0.30
0.91
0.30a
Isolate 3
0.29
0.37
0.35
1.01
0.34a*
Isolate 4
0.30
0.39
0.29
0.98
0.33a
Isolate 5
0.30
0.37
0.31
0.98
0.33a
Isolate 6
0.32
0.35
0.26
0.93
0.31a
Isolate 7
0.31
0.32
0.24
0.87
0.29ab
Isolate 8
0.30
0.31
0.23
0.84
0.28abc
Isolate 9
0.29
0.30
0.32
0.91
0.30a
Isolate 10
0.27
0.33
0.26
0.86
0.29ab
Means with the same letter are not significantly different at 5% level DMRT
22
Final Height
As table 5 shows, Isolate 8 enhanced the height of Chinese cabbagehowever
isolates2, 1, 7, 5, and 9 slightly gave different result with the control. Isolate3gave a
similar result with the controlwhereasisolate 6, 5 and 4 did not improve the height of
chinese cabbage as less result was obtained compared to the control.
Percentage Incidence of Club Root
Table 6 shows that significantnumber of club root infectionappeared under the
control fol owed by isolate 6, isolate 4 and 5, isolate 1 and 7, isolate 2, isolate 8, isolate 9
and isolate 3 then isolate 10 as the lowest. Low number of infection from isolate 10, 3
and 9 implies that the bacterial isolates inoculated in the soilcan lessen club root infection
compared other isolates. These isolates can be a good bio control agent against club root.
Table 5.Effect of the different Isolates on the final height (inches)of Chinese cabbage
taken after three months
TREATMENT
MEAN
ACTUAL
TRANSFORMED
control
6.97
2.73
isolate 1
7.87
2.89
isolate 2
8.20
2.95
isolate 3
6.97
2.73
isolate 4
4.67
2.06
isolate 5
7.13
2.76
isolate 6
5.80
2.26
isolate 7
7.63
2.85
isolate 8
8.67
3.01
isolate 9
7.40
2.80
isolate 10
7.13
2.76
Table 6.Percentage (%) incidence of club root as affected by the different isolates taken
at harvest
23
TREATMENTS
REPLICATES
TOTAL
MEAN
R
R
R
1
2
3
control
100%
100%
80%
280%
93.33%
isolate 1
60%
60%
100%
220%
73.33%
isolate 2
100%
60%
40%
200%
66.67%
isolate 3
40%
40%
40%
120%
40.00%
isolate 4
100%
80%
60%
240%
80.00%
isolate 5
80%
80%
80%
240%
80.00%
isolate 6
80%
80%
100%
260%
86.67%
isolate 7
60%
80%
80%
220%
73.33%
isolate 8
20%
100%
60%
180%
60.00%
isolate 9
40%
40%
60%
140%
46.67%
isolate 10
20%
40%
20%
80%
26.66%
Club Root Severity
Result of club root severity (Table 7) corresponds with the result of club root
incidence which means that samples with the highest incidence also has the highest
severity. Sample plants under the control showed the highest club root severity rating
fol owed by sample plants under isolate 4, 5 and 6, then isolates 1, 2 and 7, isolates 8, 3
and 9 then lowest severity is recorded under isolate 10. Reduction of club root severity in
treatments 10, 9 and 3 suggests that these bacterial isolates applied to the soil can
suppress club root development.
Table 7. Club root severity as affected by the different isolates, taken at harvest
24
TREATMENTS
REPLICATIONS
TOTAL
MEAN
R
R
R
1
2
3
Control
a
6
5
5
16
5.33
Isolate 1
abc
3
4
5
12
4.00
Isolate 2
abc
5
4
2
11
3.67
Isolate 3
c
3
3
2
08
2.67
Isolate 4
ab
6
4
4
14
4.67
Isolate 5
ab
5
5
4
14
4.67
Isolate 6
ab
4
4
6
14
4.67
Isolate 7
abc
3
4
4
11
3.67
Isolate 8
bc
2
4
3
09
3.00
Isolate 9
c
3
3
2
08
2.67
Isolate 10
c
2
3
2
07
2.33
Means with the same letter are not significantly different at 5% level DMRT
Fresh Weight of Vegetitave Parts
Among al isolates,isolate 1provides the highest weight (Table 8). Since better
result was obtained compared with control, the result implies that isolate 1 can enhanced
the vegetation of c. cabbage. Isolate 8 is the second highest fol owed by isolate 2, 3
then7. Isolate 4 has the lowest weight as maybe caused by the early death of some sample
plants due to severe club root infection.Figure 1 shows the sample plant from treatment 4
that was severely infected with club root.
25
Figure 13. Sample plant
severely infected
by club root
Table 8.Fresh weights of vegetitave parts (g) taken at harvest
TREATMENT
MEAN
ACTUAL
TRANSFORMED
Control
27.33
5.07bc
Isolate 1
100.0
9.99a
Isolate 2
61.00
7.72abc
Isolate 3
57.67
7.50abc
Isolate 4
17.00
3.62c
Isolate 5
32.67
5.66abc
Isolate 6
50.00
6.03abc
Isolate 7
53.67
7.35abc
Isolate 8
73.67
8.23ab
Isolate 9
50.67
6.84abc
Isolate 10
44.67
6.71abc
Means with the same letter are not significantly different at 5% level DMRT
26
Dry Weight of Vegetitave Parts (g)
The aerial dry weight of Chinese cabbage was very significant under isolate1
(Table 9). This result shows that isolate 1 is a good plant growth promoting bacteria since
it gave the highest dry weight. Isolate2, 3, and 7 and isolate 8gives result that is similar
with the control. Whereas isolate4, 5, 6, 9 and 10provide lower weight that was obtained
under the control.
Other Observations
Severe attack (Figure 14 and 15 ) of insects during the conduct of the experiment
affected the vegetative part of the plants including the quality of the harvest. Incidence of
cabbage butterfly larvae was high at the start of the study. Cultural practice was
implemented however at the later part of the study, severe attack of diamond back moth
larvae occurred. The use of integrated pest management approach was applied but found
to be ineffective because of severe insect infestation. Use of organic approach was not
part of the option since it might interfere on the effects of the isolates.
Table 9.Dry weightof vegetative parts (g),obtained seven weeksafter air drying
TREATMENT
MEAN
ACTUAL
TRANSFORMED
Control
2.79
1.81ab
Isolate 1
5.21
2.38a
Isolate 2
2.93
1.84ab
Isolate 3
2.62
1.74ab
Isolate 4
0.75
1.25b
Isolate 5
1.52
1.43b
Isolate 6
1.98
1.48b
Isolate 7
3.21
1.93ab
Isolate 8
3.16
1.89ab
Isolate 9
2.12
1.60b
Isolate 10
1.57
1.41b
Means with the same letter are not significantly different at 5% level DMRT
27
Figure 14.Chinese cabbage sample plant
Figure 15. Skeletonized chinese cabbage
infestedby cabbage
infested bydiamond back moth
butterfly larvae
larvae
Fresh Root Weight
Statistical analysis revealed that no isolates had significant effect on the fresh root
weight of Chinese cabbage. However based on the actual data, highest weight was
obtained in the control (Table 10). Comparison of root weight with the club root severity
implies that club root severity influence root weight. The relationship between the club
root and root weight states that the higher the club root severity of roots, the higher is the
tendency of the root to gain more weight due to hypertrophy.
Table 10.Fresh weight of roots (g) taken at harvest
TREATMENT
MEAN
ACTUAL
TRANSFORMED
control
2.77
1.74
isolate 1
1.30
1.33
isolate 2
2.63
1.76
isolate 3
0.66
1.07
isolate 4
0.85
1.10
isolate 5
1.43
1.32
isolate 6
0.74
1.07
isolate 7
1.07
1.23
isolate 8
1.83
1.44
isolate 9
0.69
1.08
isolate 10
0.75
1.14
28
Root Dry Weight
Inoculated chinese cabbage with isolate 2 (Table 11) gave the highestdry root
weight and is significantly different with plants inoculated with isolate 5, 6, 3 and 4. This
observation is consistent with the result of fresh root weight. Isolate 10 which has the
lowest severity rating also has direct relationship with the root dry weight. The
relationship states thatthe lower the club root severity is, the lesser the weight of the roots
will be.
Table 11.Roots dry weight (g) recorded after seven weeks of air drying
TREATMENT
MEAN
ACTUAL
TRANSFORMED
control
ab
1.00
1.21
isolate 1
abc
0.67
1.08
isolate 2
a
0.99
1.22
isolate 3
c
0.32
0.91
isolate 4
c
0.20
0.83
isolate 5
bc
0.38
0.93
isolate 6
c
0.37
0.92
isolate 7
abc
0.53
1.01
isolate 8
abc
0.75
1.09
isolate 9
c
0.30
0.89
isolate 10
c
0.25
0.82
29
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The study, aimed to determine the most beneficial bacteria and the standard
dilution required forthe bacteria to work effectivelyin improving plant growth and
rducingclubroot severity. The seedling experiment utilized the CRD Factorial while pot
experiment fol owed CRD.
Seedling experiment revealed that six isolates from the dilution of 104 , six
isolates from the dilution of 105 and three isolates from the dilution of 106 enhanced seed
germination rate of chinese cabbage to 100%. Likewise, isolates nine and ten sigificantly
improved the hieght of seeedlings using dilutions 104,105and106.
On the other hand, pot experiment showed that isolates 3, 4, 5, 6, 1 and 9
significantly increased the nitrogen content of the soil after harvest. In addition, isolates
10, 3 and 9 gave the lowest percent clubrootinfection of 26.66 %, 40 % and 46.67%. The
same isolates gave the lowest club root severity infection of 2.3 and 2.67. In tems of
vegetative fresh weight, isolate 1 gave the highest.
From the results, some bacterial isolates and actinomycetes-like organism from
sunflower soil improved seed germination rate and plant height, enhanced nitrogen
uptake and reduced club root severity.
30
Conclusion
Some isolates showed good effect on the growth and development of chinese
cabbage and were able to reduced clubroot infection and severity. Isolates 2 and 8
enhanced the height of chinese cabbage. While isolates 1 and 8improved the vegetative
weight of chinese cabbage. Percentage (%) nitrogen content analysis also showed that
presence of isolate 1,2,3,4,5,6,9 in the soil enhanced the availability of nitrogen in the
soil.Indicative that such isolates are good biofertilizers candidates. On the other hand
Isolates 3, 9 and 10 reduced club root severity of chinese cabbage so they can be used to
manage clubroot.
Recomendations
Based on the results, the folowing are the recommendations:
1. Characterizethe bacterial isolates that has a good effect on the growth of c.
cabbage and isolates that lessened the club root severity.
2. Nitrogen analysis of sample plants must be conducted to support the result of
nitrogen analysis from soils.
3. Conduct a study to find out if combination of two or more different isolate having
different effect is applicable to attain better result.
4. Determine the link which creates antagonism of beneficial bacteria
andPlasmodiophorabrassicae.
5. Conduct field experiment utilizing the treatments.
31
LITERATURE CITED
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brassicae) in chinese cabbage. BS Thesis (Unpub.). Benguet State University, La
Trinidad, Benguet.Pp 5.
ARIFUZZAMAN, M., M. R. KHATUN, and H. RAHMAN.2010. African Journal of
Biotechnology
Vol.
9.
http://www.academicjournals.org/AJB/PDF/pdf2010/19Jul/Arifuzzaman%2
0et%20al.pdf
BANIASADI,
F.
2009.Agricultural
and
Biological
Sciences.
http://www.wadatabase.com/categories/Agricultural-and-Biological-
Sciences/
BURGE,
H.2008.
The
Environmental
Reporter.
http://www.emlab.com/s/sampling/env-report-06-2008.html
HIRSCH, A. M.2009.Brief History of the Discovery of Nitrogen-fixing Organisms.
http:/ www.mcdb.ucla.edu/Research/Hirsch/imagesb/HistoryDiscoveryN2fixingO
rganisms.pdf
IGARASHI, Y., S. MIURA, M. AZUMI, T. FURUMAI and R. YOSHIDA. 2006.
Studies on Plant-associated Actinomycetes and Their Secondary Metabolites.
http://www.pgrsa.org/2005_Proceedings/papers/034.pdf
JEFFREY, L. S. H. 2008. Isolation, Characterization and Identification of
Actinomycetesfrom
Agriculture
Soils
at
Semongok,
Sarawak.
http://ajol.info/index.php/ajb/article/viewFile/59415/47710
LINDGREN, D. T. 1992.Vegetable Garden Seed Storage and Germination
Requirements.http://www.seedman.com/veggerm.htm
NINGTHOUJAM, D.S., S. SANASAM, K. TAMREIHAO and S. NIMAICHAND.2009.
AfricanJournal of Microbiology Research Vol. 3.
http://www.academicjournals.org/ajmr/PDF/Pdf2009/Nov/Ningthoujam%20
et%20al.pdf
SHAHIDI BONJAR, G.H., P. R. FARROKHI, S. AGHIGHI, L. SHAHIDI BONJAR.
and
A.
AGHELIZADEH.2005.
Plant
Pathology
Journal.
http://docsdrive.com/pdfs/ansinet/ppj/2005/78-84.pdf
SIVAKUMAR, K. 2008. Actinomycetes.http://ocw.unu.edu/international-network-on-
water-environment-and-health/unu-inweh-course-1- mangroves/actinomycetes.pdf
32
SULASTRI, 2005.The Use of Beneficial Microorganisms in Agricultural Practices.
http://nature.berkeley.edu/BeahrsELP/Newsletter%20-
%20Summer%202009/Bacteria_in_agriculture_Sulastri.html
TITUS, A. and PEREIRA, G. N. 2005.The Role of Actinomycetes in Coffee Plantation
Ecology.http://www.ineedcoffee.com/05/actinomycetes/
WAKSMAN,
S.A.
1959.
The
Ascomycetes.
http://www.archive.org/stream/actinomycetes01waks/actino
mycetes01was_djvu.txt
WAKSMAN, S.A. and A.T. HENRICI.1943. The NomenclatureAndClassification Of
The Actinomycetes.http://jb.asm.org/cgi/reprint/46/4/337.pdf
33
APPENDICES
Appendix Table 1. Percentage germination as affected by the different isolates and the
different dilution rates taken after two weeks
TREATMENTS
REPLICATES
TOTAL
MEAN
R
R
R
1
2
3
control
100%
100%
80%
280%
93.33%
isolate 1
60%
60%
100%
220%
73.33%
isolate 2
100%
60%
40%
200%
66.67%
isolate 3
40%
40%
40%
120%
40.00%
isolate 4
100%
80%
60%
240%
80.00%
isolate 5
80%
80%
80%
240%
80.00%
isolate 6
80%
80%
100%
260%
86.67%
isolate 7
60%
80%
80%
220%
73.33%
isolate 8
20%
100%
60%
180%
60.00%
isolate 9
40%
40%
60%
140%
46.67%
isolate 10
20%
40%
20%
80%
26.66%
34
Appendix Table 2.Seedling height (in) as affected by the different isolates and the
different dilution rates measured after six weeks
TREATMENTS
REPLIICATIONS
TOTAL
MEAN
R
R
R
R
1
2
3
4
T0D0
2.53
2.23
2.69
2.58
10.03
2.51
T1D1
2.33
2.99
3.43
3.2
11.95
2.99
T1D2
4.3
3.7
3.73
4.3
16.03
4.01
T1D3
3.76
3.71
4.06
3.61
15.14
3.79
Sub-Total
10.39
10.4
11.22
11.11
43.12
10.79
T2D1
3.28
2.4
2.93
2.83
11.44
2.86
T2D2
3.1
3.18
3.16
3.49
12.93
3.23
T2D3
3
3.44
3.94
3.8
14.18
3.55
Sub-Total
9.38
9.02
10.03
10.12
38.55
9.64
T3D1
3.64
4.14
3.94
4.21
15.93
3.98
T3D2
4.19
4.04
4.46
4.35
17.04
4.26
T3D3
4.59
4.19
4.6
3.6
16.98
4.25
Sub-Total
12.42
12.37
13
12.16
49.95
12.49
T4D1
3.43
3.93
3.96
3.63
19.93
4.98
T4D2
4.08
3.8
3.76
3.29
14.93
3.73
T4D3
3.58
3.16
3.38
3.16
13.28
3.32
Sub-Total
11.09
10.89
11.1
10.08
48.14
12.03
T5D1
2.83
2.47
2.71
2.84
10.85
2.71
T5D2
3.04
3.3
3.25
3.51
13.1
3.28
T5D3
3.66
3.54
3.56
3.36
14.12
3.53
Sub-Total
9.53
9.31
9.52
9.71
38.07
9.52
T6D1
2.26
2.39
3.36
4.43
12.44
3.11
T6D2
4.33
3.88
3.97
3.86
16.04
4.01
T6D3
3.75
3.43
4.34
3.1
14.62
3.66
Sub-Total
10.34
9.7
11.67
11.39
43.1
10.78
T7D1
3.69
2.19
3.34
3.44
12.66
3.17
T7D2
3.44
3.55
3.96
3.91
14.86
3.72
T7D3
4.11
435
3.8
3.7
15.96
3.99
Sub-Total
11.24
440.74
11.1
11.05
43.48
10.88
T8D1
2.84
2.73
3.11
3.14
11.82
2.96
T8D2
3.77
3.97
3.2
3.18
14.12
3.53
T8D3
4.09
2.42
4.01
2.63
13.15
3.29
Sub-Total
10.7
9.12
10.32
8.95
39.09
9.78
T9D1
3.18
3.44
3.48
4.4
14.5
3.63
T9D2
3.96
4.66
4.66
4.41
17.69
4.42
T9D3
4.23
4.38
4.44
3.73
16.78
4.2
Sub-Total
11.37
12.48
12.58
12.54
48.97
12.25
T10D1
4.11
3.35
3.8
5.34
16.6
4.15
T10D2
4.76
5.26
5.39
4.61
20.02
5.01
T10D3
4.67
4.31
5.43
4.22
18.63
4.66
Sub-Total
13.54
12.92
14.62
14.17
55.25
13.82
35
SIMPLIFIED DATA
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
2.51
Isolate 1
2.99
4.01
3.79
3.60d
Isolate 2
2.86
3.23
3.55
3.21d
Isolate 3
3.98
4.26
4.25
4.16b
Isolate 4
4.98
3.73
3.32
4.01d
Isolate 5
2.71
3.28
3.53
3.17d
Isolate 6
3.11
4.01
3.66
3.59cd
Isolate 7
3.17
3.72
3.99
3.63d
Isolate 8
2.96
3.53
3.29
3.26d
Isolate 9
3.63
4.42
4.20
4.08bc
Isolate 10
4.15
5.01
4.66
4.61a
MEAN
3.454b
3.92a
3.824a
Means with the same letter are not significantly different at 5% level DMRT
ANALYSIS OF VARIANCE
Source of
DF
SS
MS
Fc
Probability
variance
Factor A
9
20.332
2.259
8.1971
0.0000
Factor B
2
9.808
4.904
17.7941
0.0000
AB
18
4.179
0.232
0.8424
Error
90
24.803
0.276
Total
119
59.122
Coefficient of Variation- 14.09 %
36
Appendix Table 3. Root length (in) as affected by the different isolates and the different
dilution rates measured after six weeks
Treatment/Dilution
REPLICATIONS
Total
Mean
R1
R2
R3
R4
T0D0
5.53
07.23
07.63
10.07
30.46
7.62
T1D1
7.43
09.77
10.05
12.40
39.65
9.91
T1D2
7.90
12.3
9.03
11.67
40.9
10.23
T1D3
8.03
09.9
9.70
06.10
33.73
8.43
Sub-Total
23.36
31.97
28.78
30.17
114.28
28.57
T2D1
10.43
9.27
12.7
8.2
40.6
10.15
T2D2
10.70
10.9
9.3
8.03
38.93
9.73
T2D3
11.77
9.97
13.7
8.1
43.54
10.89
Sub-Total
32.9
30.14
35.7
24.33
123.07
30.77
T3D1
8.30
8.03
9.17
7.6
15.9
8.28
T3D2
9.70
9.87
8.7
10
19.7
9.57
T3D3
11.67
9.3
8.33
9.9
21.57
9.8
Sub-Total
29.67
27.2
26.2
27.5
57.17
27.65
T4D1
7.5
13.6
12.17
9.83
43.1
10.78
T4D2
11.2
10.7
11.6
10.73
44.23
11.06
T4D3
9.93
12.6
8.37
13.03
43.93
10.98
Sub-Total
28.63
36.9
32.14
33.59
131.26
32.82
T5D1
11.13
11.87
10.53
10.63
44.16
11.04
T5D2
11.8
13.13
8.23
8.6
41.76
10.44
T5D3
10.63
13.57
11.87
10.3
46.37
11.59
Sub-Total
33.56
38.57
30.63
29.53
132.29
33.07
T6D1
9.77
11.07
11.33
10.53
42.7
10.68
T6D2
11.33
9.23
13.53
12.47
46.56
11.64
T6D3
10.63
15.47
10.77
10.33
47.2
11.8
Sub-Total
31.73
35.77
35.63
33.33
136.46
34.12
T7D1
8.77
8.57
11.4
10.1
38.84
9.71
T7D2
11.4
5.93
6.93
8.73
32.99
8.25
T7D3
11.63
14.53
7.77
8.83
42.76
10.69
Sub-Total
31.8
29.03
26.1
27.66
114.59
28.65
T8D1
10.37
8.3
14.57
10.2
43.44
10.86
T8D2
9.16
16.37
10.47
9.9
45.9
11.48
T8D3
7.54
9.23
10.63
13.53
40.93
10.23
Sub-Total
27.07
33.9
35.67
33.63
130.27
32.57
T9D1
11.63
8.3
12.93
10.27
43.13
10.78
T9D2
9.47
8.33
11.67
8.7
38.17
9.54
T9D3
7.57
8.43
8.03
17.8
41.83
10.46
Sub-Total
28.67
25.06
32.63
36.77
123.13
32.57
T10D1
10.23
10.83
11
12.1
44.16
11.04
T10D2
11.27
11.73
8.4
14.1
45.5
11.38
T10D3
11.67
10.33
13.6
9.87
45.47
11.37
Sub-Total
33.17
32.89
33
36.07
135.13
33.79
37
SIMPLIFIED DATA
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
07.62
Isolate 1
09.91
10.23
08.43
09.52
Isolate 2
10.15
09.73
10.89
10.26
Isolate 3
08.28
09.57
09.80
09.22
Isolate 4
10.78
11.06
10.98
10.94
Isolate 5
11.04
10.44
11.59
11.02
Isolate 6
10.68
11.64
11.80
11.37
Isolate 7
09.71
08.25
10.69
09.55
Isolate 8
10.86
11.48
10.23
10.86
Isolate 9
10.78
11.38
10.46
10.87
Isolate 10
11.04
09.54
11.37
10.65
MEAN
10.323
10.332
10.624
ANALYSIS OF VARIANCE
SOURCE
DF
SS
MS
Fc
Tabulated F
OF
.05
.01
VARIANCE
Factor A
9
124.983
13.887
0.9269
1.98 2.61
Factor B
2
14.110
7.055
0.4709
AB
18
192.109
10.673
0.7124
Error
90
1348.413
14.982
Total
119
1679.615
Coefficient of Variation- 35.79 %
38
Appendix Table 4. Initial and final percentage nitrogen content of soil samples, taken
before the introduction of bacteria and after harvest
TREATMENTS
REPLICATIONS
TOTAL
MEAN
R1
R2
R3
Initial samples
0.23
0.25
0.22
0.70
0.23c
T0 - control
0.25
0.24
0.23
0.72
0.24bc
T1 – isolate 1
0.30
0.32
0.31
0.93
0.31a
T2 - isolate 2
0.30
0.31
0.30
0.91
0.30a
T3 – isolate 3
0.29
0.37
0.35
1.01
0.34a*
T4 – isolate 4
0.30
0.39
0.29
0.98
0.33a
T5 – isolate 5
0.30
0.37
0.31
0.98
0.33a
T6 – isolate 6
0.32
0.35
0.26
0.93
0.31a
T7 – isolate 7
0.31
0.32
0.24
0.87
0.29ab
T8 – isolate 8
0.30
0.31
0.23
0.84
0.28abc
T9 – isolate 9
0.29
0.30
0.32
0.91
0.30a
T10 – isolate 10
0.27
0.33
0.26
0.86
0.29ab
Grand Total
10.640
Grand mean
0.296
ANALYSIS OF VARIANCE
SOURCE OF DF
SS
MS
Fc
Tabulated F
VARIANCE
.05
.01
Treatments
11
0.034
0.003
3.000*
2.22
3.09
Error
24
0.028
0.001
Total
35
0.063
Coefficient of variation = 8.95
*= Significant at 5% level of significance
39
Appendix Table 5. Effect of the different Isolates on the final height (inches) of Chinese
cabbage taken after three months
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
6.5
6.4
8.0
20.90
6.97
T1 – isolate 1
7.1
8.5
8.0
23.60
7.87
T2 - isolate 2
8.5
8.6
7.5
24.60
8.20
T3 – isolate 3
6.8
6.6
7.5
20.90
6.97
T4 – isolate 4
0.0
6.5
7.5
14.0
4.67
T5 – isolate 5
6.5
6.6
8.3
21.40
7.13
T6 – isolate 6
8.9
8.5
0.0
17.40
5.80
T7 – isolate 7
7.6
6.7
8.6
22.90
7.63
T8 – isolate 8
6.0
11
9.0
26.00
8.67
T9 – isolate 9
7.0
6.5
8.7
22.20
7.40
T10 – isolate 10
6.5
7.9
7.0
21.40
7.13
TRANSFORMED DATA
TREATMENTS RIPLICATIONS
TOTAL
MEAN
R1
R2
R3
T0 - control
2.65
2.62
2.92
8.19
2.73
T1 – isolate 1
2.75
3.00
2.92
8.67
2.89
T2 - isolate 2
3
3.02
2.83
8.85
2.95
T3 – isolate 3
2.70
2.66
2.83
8.19
2.73
T4 – isolate 4
0.71
2.65
2.83
6.19
2.06
T5 – isolate 5
2.65
2.66
2.97
8.28
2.76
T6 – isolate 6
3.07
3.00
0.71
6.78
2.26
T7 – isolate 7
2.85
2.68
3.02
8.55
2.85
T8 – isolate 8
2.55
3.39
3.08
9.02
3.01
T9 – isolate 9
2.73
2.65
3.03
8.41
2.80
T10 – isolate 10 2.65
2.90
2.74
8.29
2.76
40
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
2.499
0.2499
0.78
0.6510
TRT
10
2.499
0.2499
0.78
0.6510
Error
32
7.092
0.32223
Corrected Total 32
9.591
Coefficient of Variation = 20.95280%
Appendix Table 6. Percentage incidence of club root as affected by the different isolates
taken at harvest
TREATMENTS
REPLICATIONS
Total
mean
R
R
R
1
2
3
T0 - control
100%
100%
80%
280%
93.33%
T1 – isolate 1
60%
60%
100%
220%
73.33%
T2 - isolate 2
100%
60%
40%
200%
66.67%
T3 – isolate 3
40%
40%
40%
120%
40%
T4 – isolate 4
100%
80%
60%
240%
80%
T5 – isolate 5
80%
80%
80%
240%
80%
T6 – isolate 6
80%
80%
100%
260%
86.67%
T7 – isolate 7
60%
80%
80%
220%
73.33%
T8 – isolate 8
20%
100%
60%
180%
60%
T9 – isolate 9
40%
40%
60%
140%
46.67%
T10 – isolate 10
20%
40%
20%
80%
26.66%
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
12921.21212
1292.12121
3.44
0.0075
TRT
10
12921.21212
1292.12121
3.44
0.0075
Error
32
8266.66667
375.75758
Corrected Total 32
21187.87879
Coefficient of Variation = 29.34346%
41
Appendix Table 7. Club root severity as affected by the different isolates, taken at harvest
TREATMENTS
REPLICATIONS
Total
Mean
R
R
R
1
2
3
T
a
0 - control
6
5
5
16
5.33
T
abc
1 – isolate 1
3
4
5
12
4
T
abc
2 - isolate 2
5
4
2
11
3.67
T
c
3 – isolate 3
3
3
2
8
2.67
T
ab
4 – isolate 4
6
4
4
14
4.67
T
ab
5 – isolate 5
5
5
4
14
4.67
T
ab
6 – isolate 6
4
4
6
14
4.67
T
abc
7 – isolate 7
3
4
4
11
3.67
T
bc
8 – isolate 8
2
4
3
9
3
T
c
9 – isolate 9
3
3
2
8
2.67
T
c
10 – isolate 10
2
3
2
7
2.33
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
30.061
3.0061
3.67
0.0052
TRT
10
30.061
3.0061
3.67
0.0052
Error
32
18.00
0.8182
Corrected Total 32
48.061
Coefficient of Variation = 24.07228%
42
Appendix Table 8. Fresh weight of vegetative parts (g) taken at harvest
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
50
13
19
82.0
27.33
T1 – isolate 1
82
123
95
300
100
T2 - isolate 2
35
88
60
183
61.00
T3 – isolate 3
50
36
87
173
57.67
T4 – isolate 4
0.0
21
30
51.0
17.00
T5 – isolate 5
31
19
48
98.0
32.67
T6 – isolate 6
81
69
0.0
150
50.00
T7 – isolate 7
59
45
57
161
53.67
T8 – isolate 8
30
135
56
221
73.67
T9 – isolate 9
53
16
83
152
50.67
T10 – isolate 10
40
47
47
134
44.67
43
TRANSFORMED DATA
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
7.11
3.67
4.42
15.2
5.07
T1 – isolate 1
9.08
11.11
9.77
29.96
9.99
T2 - isolate 2
5.96
9.41
7.78
23.15
7.72
T3 – isolate 3
7.11
6.04
9.35
22.5
7.5
T4 – isolate 4
0.71
4.64
5.52
10.87
3.62
T5 – isolate 5
5.61
4.42
6.96
16.99
5.66
T6 – isolate 6
9.03
8.34
0.71
18.08
6.03
T7 – isolate 7
7.71
6.75
7.58
22.04
7.35
T8 – isolate 8
5.52
11.64
7.52
24.68
8.23
T9 – isolate 9
7.31
4.06
9.14
20.51
6.84
T10 – isolate 10 6.36
6.89
6.89
20.14
6.71
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
86.4394
8.6439
3.69
0.1474
TRT
10
86.4394
8.6439
3.69
0.1474
Error
32
122.7707
5.1259
Corrected Total 32
199.2100
Coefficient of Variation = 33.33650%
44
Appendix Table 9. Dry weight of vegetative parts (g), obtained seven weeks after air
drying
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
3.41
3.06
1.89
8.36
2.79
T1 – isolate 1
5.01
4.72
5.91
15.64
5.21
T2 - isolate 2
2.18
4.37
2.24
8.79
2.93
T3 – isolate 3
2.70
1.26
3.89
7.85
2.62
T4 – isolate 4
0.00
0.90
1.36
2.26
0.75
T5 – isolate 5
1.30
1.16
2.11
4.57
1.52
T6 – isolate 6
3.51
2.44
0.00
5.95
1.98
T7 – isolate 7
3.17
3.08
3.37
9.62
3.21
T8 – isolate 8
2.01
4.68
2.78
9.47
3.16
T9 – isolate 9
2.34
0.96
3.06
6.36
2.12
T10 – isolate 10 1.99
0.58
2.13
4.70
1.57
TRANSFORMED DATA
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
1.98
1.89
1.55
5.42
1.81
T1 – isolate 1
2.35
2.28
2.53
7.16
2.38
T2 - isolate 2
1.64
2.21
1.66
5.51
1.84
T3 – isolate 3
1.79
1.33
2.10
5.22
1.74
T4 – isolate 4
0.71
1.18
1.86
3.75
1.25
T5 – isolate 5
1.34
1.29
1.62
4.25
1.42
T6 – isolate 6
2.00
1.72
0.71
4.43
1.48
T7 – isolate 7
1.92
1.89
1.97
5.78
1.93
T8 – isolate 8
1.58
2.28
1.81
5.67
1.89
T9 – isolate 9
1.69
1.21
1.89
4.79
1.60
T10 – isolate 10
1.58
1.04
1.62
4.24
1.41
45
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
3.0480
0.3048
2.23
0.0564
TRT
10
3.0480
0.3048
2.23
0.0564
Error
32
3.0103
0.1368
Corrected Total
32
6.0583
Coefficient of Variation = 21.71296%
Appendix Table 10. Fresh weight of roots (g) taken at harvest
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
4.82
0.64
2.86
8.32
2.77
T1 – isolate 1
1.01
0.96
1.92
3.89
1.30
T2 - isolate 2
2.25
1.98
3.66
7.89
2.63
T3 – isolate 3
0.40
0.58
1.01
1.99
0.66
T4 – isolate 4
0
0.57
1.97
2.54
0.85
T5 – isolate 5
3.15
0.25
0.89
4.29
1.43
T6 – isolate 6
1.64
0.59
0
2.23
0.74
T7 – isolate 7
0.62
0.59
2.01
3.22
1.07
T8 – isolate 8
0.43
4.07
1
5.5
1.83
T9 – isolate 9
0.48
0.35
1.25
2.08
0.69
T10 – isolate 10
1.12
0.67
0.45
2.24
0.75
TRANSFORMED DATA
TREATMENTS
REPLICATION
Total
mean
R1
R2
R3
T0 - control
2.31
1.07
1.83
5.21
1.74
T1 – isolate 1
1.23
1.21
1.56
4
1.33
T2 - isolate 2
1.66
1.57
2.04
5.27
1.76
T
3 – isolate 3
0.95
1.04
1.23
3.22
1.07
T4 – isolate 4
0.71
1.03
1.57
3.31
1.10
T5 – isolate 5
1.91
0.87
1.18
3.96
1.32
T6 – isolate 6
1.46
1.04
0.71
3.21
1.07
T7 – isolate 7
1.06
1.04
1.58
3.68
1.23
T8 – isolate 8
0.96
2.14
1.22
4.32
1.44
T9 – isolate 9
0.99
0.92
1.32
3.23
1.08
T10 – isolate 10
1.27
1.17
0.98
3.42
1.14
46
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
1.9323
0.19323
1.27
0.3048
TRT
10
1.9323
0.19323
1.27
0.3048
Error
32
3.3461
0.1521
Corrected Total 32
5.2784
Coefficient of Variation = 30.04843%
Appendix Table 11.Dry weight of Roots (g) recorded after seven weeks of air drying
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
0.98
0.43
1.58
3
1
T1 – isolate 1
0.74
0.45
0.82
2.01
0.67
T2 - isolate 2
0.97
0.90
1.09
2.96
0.99
T3 – isolate 3
0.28
0.36
0.33
0.97
0.32
T4 – isolate 4
0.00
0.31
0.29
0.6
0.2
T5 – isolate 5
0.46
0.12
0.55
1.13
0.38
T6 – isolate 6
0.59
0.51
0
1.1
0.37
T7 – isolate 7
0.32
0.42
0.84
1.58
0.53
T8 – isolate 8
0.35
1.46
0.43
2.24
0.75
T9 – isolate 9
0.11
0.24
0.55
0.9
0.3
T10 – isolate 10 0.19
0.32
0.24
0.75
0.25
47
TRANSFORMED DATA
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
1.22
0.96
1.44
3.62
1.21
T
1 – isolate 1
1.11
0.97
1.15
3.23
1.08
T2 - isolate 2
1.21
1.18
1.26
3.65
1.22
T3 – isolate 3
0.88
0.93
0.91
2.72
0.91
T4 – isolate 4
0.71
0.9
0.89
2.5
0.83
T5 – isolate 5
0.98
0.79
1.02
2.79
0.93
T6 – isolate 6
1.04
1
0.71
2.75
0.92
T7 – isolate 7
0.91
0.96
1.16
3.03
1.01
T8 – isolate 8
0.92
1.4
0.96
3.28
1.09
T9 – isolate 9
0.78
0.86
1.02
2.66
0.89
T10 – isolate 10
0.69
0.91
0.86
2.46
0.82
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
0.5909
0.05909
2.66
0.0269
TRT
10
0.5909
0.05909
2.66
0.0269
Error
32
0.4891
0.02223
Corrected Total 32
1.07999
Coefficient of Variation = 15.05225%
AGAPITO, EMERLOU P. APRIL 2012.Evaluation of Bacteria and Actinomycetes-like
Organisms against Club Root (PlasmodiophoraBrassicae) in Chinese Cabbage
(BrassicaPekinensis). Benguet State University, La Trinidad, Benguet.
Adviser: Asuncion L. Nagpala. PhD.
ABSTRACT
The study was conducted at the department of plant pathology green house, Benguet
State University, LaTrinidad, Benguet from April 2011to January 2012to evaluate the effect of
bacterial isolates and actinomycetes-like organismson the growth and development of Chinese
cabbage and their effects on the incidence and severity of club root.
Seedling experiment revealed thatsix isolates from the dilution of 104, six isolates from
the dilution of 105and three isolates from the dilution of 106enhanced seed germination rate of
chinese cabbage to 100%. Likewise, isolates 9 and 10sigificantly improved the height of
seedlings using dilutions 104,105and106.
On the other hand, pot experiment showed that isolates 3, 4, 5,6,1 and 9 significantly
increased the nitrogen content of the soil after harvest. In addition, isolates 10, 3 and 9 gave the
lowest percent club root infection of 26.66 %, 40 % and 46.67%. The same isolates gave the
lowest club root severity infection of 2.3 % and 2.67 %. In terms of vegetative fresh weight,
isolate 1 gave the highest.
From the results, some bacterial isolates and actinomycetes-like organism from sunflower
soil improved seed germination rate and plant height, enhanced nitrogen uptake and reduced club
root severity.
Evaluation of Bacteria and Actinomycetes‐like Organisms against Club Root (PlasmodiophoraBrassicae) in
Chinese Cabbage (BrassicaPekinensis) / Emerlou P. Agapito. 2012
TABLE OF CONTENTS
Page
Bibliograpy…………………………………………………………………….......... i
Abstract……………………………………………………………………………… i
Table of Contents……………………………………………………………………
ii
INTRODUCTION……………………………………………………………………. 1
REVIEW OF LITERATURE…………………………………………………………
4
The Nomenclature and Classification
of the Actinomycetes…………………………………………………………
4
Isolation, Identification, Cultivation
and Preservation……………………………………………………………...
5
Nitrogen Fixation……………………………………………………………..
7
Activity of Actinomycetes
upon Plant Pathogenic Fungi……………………………………………......
8
Causation of Plant Diseases………………………………………………….
8
Plant Growth Promotion Activity
ofSecondary Metabolites………………………………………………………
9
MATERIALS AND METHODS……………………………………………………..
10
Seedling Experiment…………………………………………………………
10
Pot
Experiment………………………………………………………………..
11
Data Gathered…………………………………………………………………
13
RESULTS AND DISCUSSION……………………………………………………… 15
Seedling Experiment…………………………………………………………………... 15
Percentage Seed Germination
asAffected by the Different isolates……………………………………….......
15
Percentage Seed Germination
asAffectedby the Different Dilution Rates……………………………………
15
Interaction Effect on the Seed
15
Germination…………………………………………………………………..
Seedlings Height as Affected
16
bythe Different Isolates………………………………………………………
Seedlings Height as Affected
17
by the Different Dilution Rates……………………………………………….
Interaction
Effect
17
on the Seedlings Height………………………………………………………
Seedlings Root Length as Affected
bythe Different Isolates………………………………………………………
19
Seedlings Root Length as Affected
by the Different Dilution Rates………………………………………………
19
Interaction
Effect on the Seedlings
Root Length…………………………………………………………………..
20
Pot experiment…………………………………………………………………………
21
Nitrogen Content of the Soil………………………………………………..
21
Final
Height……………………………………………………………………
22
Percentage Incidence of Club root…………………………………………….
22
Club Root Severity……………………………………………………………
23
Fresh Weights of Vegetative Parts……………………………………………..
24
Dry Weights of Vegetative Parts……………………………………………… 26
Fresh Roots Weight…………………………………………………………..
27
Roots Dry Weight……………………………………………………………
28
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary……………………………………………………………………… 29
Conclusion ……………………………………………………………………
30
Recommendations……………………………………………………………. 31
LITERATURE CITED………………………………………………………………..
31
APPENDICES……………………………………………………………………….. 33
1
INTRODUCTION
Non adverse effects on the environment of biocontrol strategies of pest
management are priorities of tomorrow’s world agriculture (Baniasadi, 2009). Biological
control is slow but can be long lasting, inexpensive, and harmless to living organisms and
the ecosystem. It neither eliminates the pathogen nor the disease, but brings them into
natural balance. Intensive research on plant growth promoting bacteria (PGPB) is
underway worldwide for developing biofertilizers and biocontrol agents (BCAs) as better
alternatives to chemicals (Ningthoujam, 2009). High input agriculture is increasingly
recognized as contributing to the degradation of environment and health besides
demanding high costs due to its dependence on chemical inputs (Sulastri, 2005).
Biocontrol with beneficial bacteria is one promising alternative to fungicides.
Hydrolases such as chitinase contribute to degradation of fungal cel wal s. Chitin is the
second most abundant polysaccharide in nature and a major component of fungal walls,
insect exoskeletons and crustacean shells. Chitinase secreted by biological control agents
is likely to be effective against pathogenic fungi which cel wal s are mainly made up of
chitin (Ningthoujam, 2009).Actinomycetes are active biocontrol agents due to their
antagonistic properties against wide range of plant pathogenic fungi (Baniasadi, 2009).
This group of microorganisms is best known for their ability to produce bio-active
metabolites including antibiotics, plant growth factors, and other substances. Many of the
presently used antibiotics such as streptomycin, gentamicin, rifamycin and erythromycin
are the product of Actinomycetes (Jeffrey, 2008).
Evaluation of Bacteria and Actinomycetes-like Organisms against Club Root
(PlasmodiophoraBrassicae) in Chinese Cabbage (BrassicaPekinensis) / Emerlou P. Agapito. 2012
2
Among Actinomycetes, the Streptomycetes are the dominant. The
non‐streptomycetes are called rare Actinomycetes, comprising approximately 100 genera
(Sivakumar, 2008). Streptomycesand other Actionmycetes are major contributors to
biological buffering of soils and have roles in organic matter decomposition conducive to
crop production.
Actinomycetesare responsible for much of the digestion of resistant carbohydrates
such as chitin and cellulose bioremediation. The number and types of Actinomycetes
present in a particular soil would be greatly influenced by geographical location such as
soil temperature, soil type, soil pH, organic matter content, cultivation, aeration and
moisture content. Some are able to grow at elevated temperatures (>50°C) and are
essential to the composting process (Burge, 2008).Population ofActinomycetes is
relatively lower than other soil microbes and contains a predominance of Streptomyces
that are tolerant to acid conditions. Arid soils of alkaline pH tend to contain fewer
Streptomyces and more of the rare genera such as Actinoplanes and Streptosporangium.
However, alkaliphilicActionmycetes will provide a valuable resource for novel products
of industrial interest, including enzymes and antimicrobial agents. Among
Actinomycetes, the Streptomyces are especially prolific. A search of the recent literature
revealed that at least 4,607 patents have been issued on Actinomycete related product and
processes. Streptomycescovers around 80% of total antibiotic product, with other genera
trailing numerical y. Micromonospora is second with less than one-tenth as many as
Streptomyces(Arifuzzaman, 2010).
3
The result that will be generated in this study whereby a new bacterial isolates
that wil be found effective as an enhancer of nutrients in the soil will contribute for the
growth and development of plants. Moreover the isolates that wil be found effective as
biological agent against club root will be added to the few existing bio control agents
used to manage the disease.
This study aimed to:
1. determine the effects of bacteria and Actinomycetes-like organisms on the
growth and development of chinese cabbage, and
2. determine their effects on the incidence and development of club root on
Chinese cabbage.
This study was conducted at the department of plant pathology green house,
Col ege of Agriculture, Benguet State University, La Trinidad, Benguet from April 2011
to January 2012.
4
REVIEW OF LITERATURE
The Nomenclatureand Classification
of theActinomycetes
Family Streptomycetaceae.Actinomycetes with branched slender mycelium that is
rarely or not septate forming spores on aerial hyphae and not fragmenting intooidia.
There are two genera, Streptomyces and Micromonospora(Waksman and Henrici, 1943)
Genus Streptomyces.Streptomycetaceae forming spores in chains on aerialhyphae
cal ed sporosphores. Sporosphores are apparently endogenous in origin, formed by a
segregationof protoplasm within the hypha into a series of round, oval or cylindric
bodies.Chains of spores are often spirally coiled. Sporophores may be simple orbranched.
The type of species of this newly-named genus is Streptomyces albus (Rossi-Doria
emend Krainsky) comb. nov. This species was formerly known as
ActinomycesalbusKrainsky and first described as StreptothrixalbaRossi-Doria. This is
one of the most common and best known species of the group. It is colorless with white
aerial mycelium, forming ovoidal spores in coiled chains on lateral branches of the aerial
hyphae. It is proteolytic, liquefying gelatin and peptonizing milk with the production of
alkaline reaction in the latter. It does not produce any soluble pigment eitheron an organic
or synthetic medium, but does produce a characteristic earthy ormusty odor (Waksman
and Henrici, 1943).
5
Genus Micromonospora.The name MicromonosporaOrskov, apply to those forms
which producing single conidia on lateral branches. Tsiklinskyhad previously applied the
name Thermoactinomyces to species of thisgroup, whose identity is clear from
photomicrographs. But in her descriptionof the genus she also included thermophilic
species with catenulate spores, basingthe genus on temperature relations rather on
morphology (Waksman, 1959).
Isolation, Identification, Cultivation,
and Preservation
Most of the techniques used in the isolation and cultivation of bacteria and
fungi also apply to Actinomycetes. The isolation of these organisms from soils and other
natural substrates is brought about by first plating out such materials in proper dilutions
on suitable agar or gelatin media. The plates are incubated at favorable temperatures, for
2 to 7 days, and the colonies picked and transferred to sterile liquid or solid media for
further development. A colony of an Actinomycetediffers from a bacterial colony due to
the presence of a filamentous extension of the original cell or cel s, spores, and
degradation products. It is not an accumulation of cel s originating from one or more
similar cells. These are compact, often leathery, giving a conical appearance, and have a
drysurface. According to Titus and Pereira (2005) the leathery or powdery appearance of
Actinomycetes colonies is due to the production of conidia. These are often covered with
aerial mycelium. When grown in liquid culture, either in a stationary or in a submerged
condition, the majority of Actinomycetes, notably members of the genera Streptomyces
and Micromonospora, grow in the form of flakes or spherical compact masses, leaving
the medium clear. The mass of growth can easily be removed by filtration through
6
ordinary paper. Only when growth undergoes lysis do the cellsdisintegrate completely
and a certain degree of turbidity occurs (Waksman, 1959).
The methods of studying the Actinomycetes population of soil, water, compost,
and other materials include microscopic observations,plate culture studies,and selective
culture procedures. Primarily for characterization and identification purposes,a standard
media, comprising both synthetic and organic, are most essential. Synthetic, chiefly
inorganic, media have found extensive application in the study of the morphology,
physiology, and cultural characterization of these organisms. Organic media are used for
obtaining supplementary evidence of a cultural nature, especially for strains that do not
grow at al or grow only very quickly on the common inorganic media. A Media used
primarily for obtaining maximum growth, especially for the maximum production of
certain chemical substances, such as antibiotics, vitamins, or enzymes are usually
complex in composition, utilizing plant and animal materials directly or after preliminary
enzymatic or acid digestion. For maintaining cultures of Actinomycetes in such a manner
as to reduce, to a minimum, degeneration and variationof the culture a suitable media,
comprising both artificial and natural, such as sterile soil, and suitable conditions of
growth thus make possible for the preservation of type cultures for comparative purposes.
The great majority of Actinomycetes are aerobic and very few are anaerobic and many
are microaerophilic. To supply proper aeration, the organisms are grown on the surface of
solid media, or in shallow liquid layers, or in a thoroughly aerated submerged condition.
For anaerobic growth, special procedures are required. Temperatures of 25-30° C are
usual y used for incubation of the great majority of Steptomyces, Nocardias, and
7
Micromonosporas. Pathogenic organisms require 37° C, and thermopiles usually require
50-60° C (Waksman, 1959).
Nitrogen Fixation
Various reports have been made in the past of the ability of one or more
Actinomycetes to fix atmospheric nitrogen. Meyen first observed nodules on alder roots
whichwas confirmed by Woronin. Brunchorst named the microbe microbes inside the
nodulesFrankiasubtilis and Hiltner recognized the nodule inhabitant as an actinomycete,
gram-positive bacteria closely related to Streptomyces. Like Streptomyces, Frankiaforms
spores, but it also produces structures known as vesiclesthat sequester the oxygen-labile
enzyme nitrogenase. The vesicle cell walls are composed ofhopanoid lipids, making them
impervious to oxygen. Itappears phase-bright under phasecontrast microscopy. Several
different Frankiastrains were alsoisolated from actinorhizal plants, including Casuarina,
Elaeagnus,and Myrica. Other Actinomycetes were also being isolated from the nodules
of diverse actinorhizalplants, but not much attention was being paid to them. In the late
1980’s, several Actinomyceteshad been isolated from nodules of Casuarinatrees
(indigenous to Australia) growing in Mexico (Hirsch, 2009).
twonocardias, N. calcarca and N. cdlulans, isolated from grassland lime soils
were found to have the capacity to fix atmospheric nitrogen to the extent of 2.0 to 4.5 mg
of X/gm of glucose or other carbon source in the medium. The second culture was also
capable of decomposing cel ulose, the amount of nitrogen fixed being 5 to 12 mg of
X/gm of cellulose decomposed (Waksman, 1959).
8
Activity of Actinomycetes
upon Plant Pathogenic Fungi
An extensive literature has accumulated upon the antagonistic effects of
Actinomycetes upon fungi, especially upon plant pathogens. Winterpresented further
evidence concerning the ability of various Actinomycetes to attack Ophiobolusgraminus,
an important parasite that attacks wheat. Sanford and Cormack tested the effect of eight
cultures of Actinomycetes upon the disease-producing fungus Helminthosporiumsativum.
In comparison with a disease rating of 66 percent for the untreated pathogen, four
Actinomycetes suppressed the virulence of the pathogen to 33, 22, and 1 per cent,
respectively; two had no marked effect; and the other two appeared to increase the
virulence by 12 and 16 percent, respectively. Perrault demonstrated that the growth of
Colletotrichumsepedonicum in agar media was impeded by several microorganisms
isolated from potato tubers affected with ring rot. Four of these organisms were
Actinomycetes and were able to produce antibiotic substances that diffused readily
through the medium and prevented al growth of the pathogen. One culture produced a
lysis of the plant pathogen (Waksman, 1959). In 2005 plant pathology journal, 10 isolates
of Actinomycetes was reported to have an antagonistic reaction to a single isolate of
Alternariasolani through agar plate method (ShahidiBonjar, 2005).
Causation of Plant Diseases
In spite of the great importance of Actinomycetes in nature, especially in the
soil, the number of plants attacked by these, as compared to the number of plants attacked
by bacteria, fungi, and viruses, is rather limited. Two species of plant which is the Irish
potato and the sugar beet plants are known to be infected by Actinomycetes and causes
9
scab. In Hoffmann’s work, numerous infection experiments were carried out with twenty
Streptomycesspecies, using a number of scab-susceptible potato varieties in the
greenhouse and under field conditions. He found out thatS. scabies was the pathogen of
potato scab and the same was true with beet scab (Waksman, 1959).
Plant growth Promotion Activity
of Secondary Metabolites
Although Actinomycetes produce numerous kinds of secondary metabolites, their
plant bioactivity is known very little. In their recent studies,Igarashi et al. (2006) have
identified at least 10 chemical y different classes of secondary metabolites produced by
Streptomyces hygroscopicus. Of these compounds, pteridic acid A induced the
adventitious root formation of kidney bean hypocotyls and growth promotion of tobacco
BY-2 cells which suggest the possible involvement of secondary metabolites in plant
growth promotion.
10
MATERIALS AND METHOD
Sterilized forest soil obtained at the forest area of Long long, Puguis, la Trinidad,
Benguet was used in the entire experiment.The sterilization period using steam pressured
drum lasted for 8 hours.
Seedling Experiment
Sterilized soil was distributed in a seedling tray with 3 x 13 holes. Different
dilutions of 104, 105 and 106 of the different isolates were prepared and 3ml each was
inoculated in seedling trays.The control was inoculated with water only. The experiment
set up fol owed the Complete Randomized Factorial Design (CRD Factorial) and was
replicated four times with 34 sample plants per treatment per replicate. Chinese cabbage
seeds were sown two weeks after inoculation. The treatments are shown below.
Treatments:
Factor A - Isolates
T0 -no bio-control added
Factor B – Dilution Rates
T1 - bacterial isolate 1
T2 - bacterial isolate 2
T3- bacterial isolate 3
T4- bacterial isolate 4
T5 - bacterial isolate 5
T6 - bacterial isolate 6
T7 -bacterial isolate 7
T8-bacterial isolate 8
T9-bacterial isolate 9
T10-bacterial isolate 10
11
Pot Experiment
The dilution of the different isolates that showed good performance during the
seedling experiment was further evaluated in a pot experiment. An amount of six kg of
soil were filled in pots measuring 6x6x11 inches and were inoculated with 30 ml 105
(best dilution) of the different isolates. Two weeks after the introduction of bacterial
isolates, every pot was infested with 5mlPlasmodiophorabrassicaehaving
sporeconcentration of 1x106 per ml.Seedlings that were taken from the seedling
experiment were transplanted in the inoculated pots one week after infestation. The
experiment set up utilized the complete randomized design (CRD) with 3 replicates
having 5 sample plants per treatment per replicate. The treatments are described below
while figure 1 shows the isolates used:
Treatments:
T0 -no bio-control added
T1 - bacterial isolate 1
T2 - bacterial isolate 2
T3- bacterial isolate 3
T4- bacterial isolate 4
T5 - bacterial isolate 5
T6 - bacterial isolate 6
T7 -bacterial isolate 7
T8-bacterial isolate 8
T9-bacterial isolate 9
T10-bacterial isolate 10
12
Figure 1. Colonies of the bacterial isolates used in the seedling
and pot experiment
13
Data Gathered:
A. Seedling experiment
1. Percentage seed germination. Germinated seeds was counted two weeks
aftersowing. Percent germination was determined using the formula,
no.f germinated seds
no.f seds own x 10
2. Seedlingheight. Height (inches) of al the 34 samples seedlings per replicate were
measured after six weeks.
3. Seedling root length. Roots of three sample plants were uprooted randomly were
measured.
B. Pot experiment
1. Nitrogen analysis of the soil. Soil samples of each replicate of the different
treatment weighing 200 grams was col ected after which was mix to come up with a
composite soil sample. The percentage N content(%N) of the soil wasestimated by
determining the organic matter content (%OM) of the soil multiplied by 0.05(%N=
%OM x 0.05). Soil analysis was done before the inoculation of bacterial isolateand after
harvest.
2. Final Height. Plant height of five samples was measured in inches one day before
theharvest.
3. Club Root Incidence. Plants infected with of club root was counted at harvest and
percent club root incidence was determined using the formula:
no.f sample plants −no.f healthy plants
% Incidence =
toal no.f plants
x 100
14
4. Club root Severity. Assessment of club root severity was based on the rating scale
of Anderson et al. as stated below:
Rating
Description
1
Normal root
2
Minor lateral clubbing at 0.5 cm diameter
3
Minor lateral clubbing at 1.2 cm diameter
4
Moderate clubbing
5
Severe clubbing in the tap root
6
Root decaying due advance infestation with plant
death.
5. Fresh and dry weights of vegetative parts. Fresh weights in grams of the
vegetative part was recorded before drying, whilethe dry weight was taken seven weeks
after air drying.
6. Fresh and dry weights of below ground parts. Fresh weights in grams of the
below ground partswas recorded before drying, while the dry weight was taken seven
weeks after air drying.
15
RESULTS AND DISCUSSION
A. Seedling Experiment
Percentage Seed Germination
asAffectedby the Different Isolates
According to Lindgren (1992), the standard percentage seed germination in
chinese cabbage is 80 % under optimum temperature.Table 1showsthe result of
germination rate two weeks after sowing.
The highest germination rate of 100% was recorded under isolate 4.Isolate 1
provided the second highest germination rate of 98.95% fol owed by isolates 2, 7 and 9.
Such result indicates that these isolates can be used as soil inoculants to improve seed
germination for they gave a better rate of germination than the control (96.86 %)
Percentage Seed Germination
asAffectedby the Different Dilution Rates
The highest germination rate of 98.12% and 97.18 % was recorded from the
dilutions of 104and 105.This means that higher concentration of isolates work effectively
in improving the germination rate of Chinese cabbage.
Interaction Effect on the Seed
Germination
Interaction between the factors suggest that al isolateimproved the germination
rate of chinese cabbage using dilutions of 104and 105 when inoculated in the soil before
sowing the seeds.Isolates withthe dilution of 106hadthe least percentage germination.
16
Table 1. Percentage germination as affected by the different isolates and the
differentdilution ratesrecorded after two weeks
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
96.86%
Isolate 1
100%
100%
96.86%
98.95%
Isolate 2
100%
100%
93.75%
97.92%
Isolate 3
100%
90.66%
100%
96.89%
Isolate 4
100%
100%
100%
100%
Isolate 5
90.66%
100%
96.86%
95.84%
Isolate 6
96.86%
96.86%
93.73%
95.82%
Isolate 7
100%
100%
93.75%
97.92%
Isolate 8
96.86%
87.5%
96.86%
93.74%
Isolate 9
96.86%
96.86%
100%
97.91%
Isolate 10
100%
100%
87%
95.67%
MEAN
98.124%
97.188%
95.881%
SeedlingsHeight as Affected
bythe Different Isolates
In terms of seedling height, Table 2 revealed that soils inoculated with isolate10
gave the tal est seedlings with the average height of 13.82 inches. This wasfollowed by
sample plantsunder isolates 3,9 and 4surpassing the control which means that thesecan
significantly enhancethe seedling height of chinese cabbage. Isolates 7, 1, and 6 slightly
increase the height of chinese cabbage.
17
Seedlings Height as Affected
bythe Different Dilution Rates
statistical analysis for the effect of different dilution rates (Table 2) shows that the
best dilutions that that can enhance the seedling height were 105and 106.
Interaction Effect on the Seedlings Height
Isolates 10, 3, 9 and 4 significantly improved the growth of chinese cabbage
seedlings using any of the dilutions while. Seedlings under isolate 10 with the dilution of
105 had the highest measurement of height.
Table 2: Seedling height (inches) as affected by the different isolates and the different
dilution rates measured after six weeks
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
2.51
Isolate 1
2.99
4.01
3.79
3.60d
Isolate 2
2.86
3.23
3.55
3.21d
Isolate 3
3.98
4.26
4.25
4.16b
Isolate 4
4.98
3.73
3.32
4.01d
Isolate 5
2.71
3.28
3.53
3.17d
Isolate 6
3.11
4.01
3.66
3.59cd
Isolate 7
3.17
3.72
3.99
3.63d
Isolate 8
2.96
3.53
3.29
3.26d
Isolate 9
3.63
4.42
4.20
4.08bc
Isolate 10
4.15
5.01
4.66
4.61a
MEAN
3.454b
3.92a
3.824a
Means with the same letter are not significantly different at 5% level DMRT
18
Figures 2 to 12 show the different set-up inoculated with isolates at different
dilution rate.
Figure 2. Seedlings not inoculated with
Figure 3. Chinese cabbage seedlings inoculated
bacterial isolates (water)
with bacterial isolate 1
Figure 4. Chinese cabbage seedlings
Figure 5. Chinese cabbage seedlings inoculated
inoculated with bacterial isolate 2
with bacterial isolate 3
Figure 6. Chinese cabbage seedlings inoculated
Figure 7. Chinese cabbage seedlings inoculated
with bacterial isolate 4
with bacterial isolate 5
Figure 8. Chinese cabbage seedlings
Figure 9. Chinese cabbage seedlings inoculated
inoculated with bacterial isolate 6
with bacterial isolate 7
19
Figure 10. Chinese cabbage seedlings
inoculated with bacterial isolate 8 Figure 11. Chinese cabbage seedlings
inoculated with bacterial isolate 9
Figure 12. Chinese cabbage seedlings inoculated
with bacterial isolate 10
SeedlingsRoot Length as Affected
Bythe Different Isolates
Table 3shows that al isolates enhanced the root length of chinese cabbage
seedling by surpassing the control. Isolate 6provided the highest root length mean of
11.37fol owed by isolate 5.
SeedlingsRoot Length as Affected
by the Different Dilution Rates
Result obtained for the effect of different dilutions on the root length of chinese
cabbage is almost the same which suggest that al dilution rates has no significant effect
on the root development of chinese cabbage.
20
Interaction Effect on the Seedlings
Root Length
The interaction between two factors suggests that isolate 6 with the dilution of
105wil improve the root development of roots in terms of length giving the highest mean
of 11.64. Main while dilution of 105used for isolate 7 gave the lowest mean of 8.25.
Table 3.Root length (inches) as affected by the different isolates and the different dilution
rates measured after six weeks
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
07.62
Isolate 1
09.91
10.23
08.43
09.52
Isolate 2
10.15
09.73
10.89
10.26
Isolate 3
08.28
09.57
09.80
09.22
Isolate 4
10.78
11.06
10.98
10.94
Isolate 5
11.04
10.44
11.59
11.02
Isolate 6
10.68
11.64
11.80
11.37
Isolate 7
09.71
08.25
10.69
09.55
Isolate 8
10.86
11.48
10.23
10.86
Isolate 9
10.78
11.38
10.46
10.87
Isolate 10
11.04
09.54
11.37
10.65
MEAN
10.32
10.33
10.62
21
B. Pot Experiment
Based on the result of seedling experiment, al isolates has a good effect on the
growth and development of chinese cabbage seedling. Using different rate of dilutions,
isolates with the dilution of 105 showed the most significantresult. This served as the
standard dilution rate for al isolate that was evaluated in the pot experiment.
Nitrogen Content of the Soil
Based on the result of % N content analysis as shown in Table 4, nitrogen content
al soil samples that were inoculated with bacterial isolatehad increased. Initial soils
withanitrogen content of 0.23 %,had significantlyincreased to0.34 %with the inoculation
of isolate 3. On the other hand, isolate 8 slightly increase the amount of nitrogen in the
soil.Under control, no significant increase can be observed.
Table 4.Initial and final percentage nitrogen content of soil samples, taken before the
inoculation of bacterial isolatesand after harvest
TREATMENT
REPLICATES
TOTAL
MEAN
S
R1
R2
R3
Initial soil
0.23
0.25
0.22
0.70
0.23c
Control
0.25
0.24
0.23
0.72
0.24bc
Isolate 1
0.30
0.32
0.31
0.93
0.31a
Isolate 2
0.30
0.31
0.30
0.91
0.30a
Isolate 3
0.29
0.37
0.35
1.01
0.34a*
Isolate 4
0.30
0.39
0.29
0.98
0.33a
Isolate 5
0.30
0.37
0.31
0.98
0.33a
Isolate 6
0.32
0.35
0.26
0.93
0.31a
Isolate 7
0.31
0.32
0.24
0.87
0.29ab
Isolate 8
0.30
0.31
0.23
0.84
0.28abc
Isolate 9
0.29
0.30
0.32
0.91
0.30a
Isolate 10
0.27
0.33
0.26
0.86
0.29ab
Means with the same letter are not significantly different at 5% level DMRT
22
Final Height
As table 5 shows, Isolate 8 enhanced the height of Chinese cabbagehowever
isolates2, 1, 7, 5, and 9 slightly gave different result with the control. Isolate3gave a
similar result with the controlwhereasisolate 6, 5 and 4 did not improve the height of
chinese cabbage as less result was obtained compared to the control.
Percentage Incidence of Club Root
Table 6 shows that significantnumber of club root infectionappeared under the
control fol owed by isolate 6, isolate 4 and 5, isolate 1 and 7, isolate 2, isolate 8, isolate 9
and isolate 3 then isolate 10 as the lowest. Low number of infection from isolate 10, 3
and 9 implies that the bacterial isolates inoculated in the soilcan lessen club root infection
compared other isolates. These isolates can be a good bio control agent against club root.
Table 5.Effect of the different Isolates on the final height (inches)of Chinese cabbage
taken after three months
TREATMENT
MEAN
ACTUAL
TRANSFORMED
control
6.97
2.73
isolate 1
7.87
2.89
isolate 2
8.20
2.95
isolate 3
6.97
2.73
isolate 4
4.67
2.06
isolate 5
7.13
2.76
isolate 6
5.80
2.26
isolate 7
7.63
2.85
isolate 8
8.67
3.01
isolate 9
7.40
2.80
isolate 10
7.13
2.76
Table 6.Percentage (%) incidence of club root as affected by the different isolates taken
at harvest
23
TREATMENTS
REPLICATES
TOTAL
MEAN
R
R
R
1
2
3
control
100%
100%
80%
280%
93.33%
isolate 1
60%
60%
100%
220%
73.33%
isolate 2
100%
60%
40%
200%
66.67%
isolate 3
40%
40%
40%
120%
40.00%
isolate 4
100%
80%
60%
240%
80.00%
isolate 5
80%
80%
80%
240%
80.00%
isolate 6
80%
80%
100%
260%
86.67%
isolate 7
60%
80%
80%
220%
73.33%
isolate 8
20%
100%
60%
180%
60.00%
isolate 9
40%
40%
60%
140%
46.67%
isolate 10
20%
40%
20%
80%
26.66%
Club Root Severity
Result of club root severity (Table 7) corresponds with the result of club root
incidence which means that samples with the highest incidence also has the highest
severity. Sample plants under the control showed the highest club root severity rating
fol owed by sample plants under isolate 4, 5 and 6, then isolates 1, 2 and 7, isolates 8, 3
and 9 then lowest severity is recorded under isolate 10. Reduction of club root severity in
treatments 10, 9 and 3 suggests that these bacterial isolates applied to the soil can
suppress club root development.
Table 7. Club root severity as affected by the different isolates, taken at harvest
24
TREATMENTS
REPLICATIONS
TOTAL
MEAN
R
R
R
1
2
3
Control
a
6
5
5
16
5.33
Isolate 1
abc
3
4
5
12
4.00
Isolate 2
abc
5
4
2
11
3.67
Isolate 3
c
3
3
2
08
2.67
Isolate 4
ab
6
4
4
14
4.67
Isolate 5
ab
5
5
4
14
4.67
Isolate 6
ab
4
4
6
14
4.67
Isolate 7
abc
3
4
4
11
3.67
Isolate 8
bc
2
4
3
09
3.00
Isolate 9
c
3
3
2
08
2.67
Isolate 10
c
2
3
2
07
2.33
Means with the same letter are not significantly different at 5% level DMRT
Fresh Weight of Vegetitave Parts
Among al isolates,isolate 1provides the highest weight (Table 8). Since better
result was obtained compared with control, the result implies that isolate 1 can enhanced
the vegetation of c. cabbage. Isolate 8 is the second highest fol owed by isolate 2, 3
then7. Isolate 4 has the lowest weight as maybe caused by the early death of some sample
plants due to severe club root infection.Figure 1 shows the sample plant from treatment 4
that was severely infected with club root.
25
Figure 13. Sample plant
severely infected
by club root
Table 8.Fresh weights of vegetitave parts (g) taken at harvest
TREATMENT
MEAN
ACTUAL
TRANSFORMED
Control
27.33
5.07bc
Isolate 1
100.0
9.99a
Isolate 2
61.00
7.72abc
Isolate 3
57.67
7.50abc
Isolate 4
17.00
3.62c
Isolate 5
32.67
5.66abc
Isolate 6
50.00
6.03abc
Isolate 7
53.67
7.35abc
Isolate 8
73.67
8.23ab
Isolate 9
50.67
6.84abc
Isolate 10
44.67
6.71abc
Means with the same letter are not significantly different at 5% level DMRT
26
Dry Weight of Vegetitave Parts (g)
The aerial dry weight of Chinese cabbage was very significant under isolate1
(Table 9). This result shows that isolate 1 is a good plant growth promoting bacteria since
it gave the highest dry weight. Isolate2, 3, and 7 and isolate 8gives result that is similar
with the control. Whereas isolate4, 5, 6, 9 and 10provide lower weight that was obtained
under the control.
Other Observations
Severe attack (Figure 14 and 15 ) of insects during the conduct of the experiment
affected the vegetative part of the plants including the quality of the harvest. Incidence of
cabbage butterfly larvae was high at the start of the study. Cultural practice was
implemented however at the later part of the study, severe attack of diamond back moth
larvae occurred. The use of integrated pest management approach was applied but found
to be ineffective because of severe insect infestation. Use of organic approach was not
part of the option since it might interfere on the effects of the isolates.
Table 9.Dry weightof vegetative parts (g),obtained seven weeksafter air drying
TREATMENT
MEAN
ACTUAL
TRANSFORMED
Control
2.79
1.81ab
Isolate 1
5.21
2.38a
Isolate 2
2.93
1.84ab
Isolate 3
2.62
1.74ab
Isolate 4
0.75
1.25b
Isolate 5
1.52
1.43b
Isolate 6
1.98
1.48b
Isolate 7
3.21
1.93ab
Isolate 8
3.16
1.89ab
Isolate 9
2.12
1.60b
Isolate 10
1.57
1.41b
Means with the same letter are not significantly different at 5% level DMRT
27
Figure 14.Chinese cabbage sample plant
Figure 15. Skeletonized chinese cabbage
infestedby cabbage
infested bydiamond back moth
butterfly larvae
larvae
Fresh Root Weight
Statistical analysis revealed that no isolates had significant effect on the fresh root
weight of Chinese cabbage. However based on the actual data, highest weight was
obtained in the control (Table 10). Comparison of root weight with the club root severity
implies that club root severity influence root weight. The relationship between the club
root and root weight states that the higher the club root severity of roots, the higher is the
tendency of the root to gain more weight due to hypertrophy.
Table 10.Fresh weight of roots (g) taken at harvest
TREATMENT
MEAN
ACTUAL
TRANSFORMED
control
2.77
1.74
isolate 1
1.30
1.33
isolate 2
2.63
1.76
isolate 3
0.66
1.07
isolate 4
0.85
1.10
isolate 5
1.43
1.32
isolate 6
0.74
1.07
isolate 7
1.07
1.23
isolate 8
1.83
1.44
isolate 9
0.69
1.08
isolate 10
0.75
1.14
28
Root Dry Weight
Inoculated chinese cabbage with isolate 2 (Table 11) gave the highestdry root
weight and is significantly different with plants inoculated with isolate 5, 6, 3 and 4. This
observation is consistent with the result of fresh root weight. Isolate 10 which has the
lowest severity rating also has direct relationship with the root dry weight. The
relationship states thatthe lower the club root severity is, the lesser the weight of the roots
will be.
Table 11.Roots dry weight (g) recorded after seven weeks of air drying
TREATMENT
MEAN
ACTUAL
TRANSFORMED
control
ab
1.00
1.21
isolate 1
abc
0.67
1.08
isolate 2
a
0.99
1.22
isolate 3
c
0.32
0.91
isolate 4
c
0.20
0.83
isolate 5
bc
0.38
0.93
isolate 6
c
0.37
0.92
isolate 7
abc
0.53
1.01
isolate 8
abc
0.75
1.09
isolate 9
c
0.30
0.89
isolate 10
c
0.25
0.82
29
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The study, aimed to determine the most beneficial bacteria and the standard
dilution required forthe bacteria to work effectivelyin improving plant growth and
rducingclubroot severity. The seedling experiment utilized the CRD Factorial while pot
experiment fol owed CRD.
Seedling experiment revealed that six isolates from the dilution of 104 , six
isolates from the dilution of 105 and three isolates from the dilution of 106 enhanced seed
germination rate of chinese cabbage to 100%. Likewise, isolates nine and ten sigificantly
improved the hieght of seeedlings using dilutions 104,105and106.
On the other hand, pot experiment showed that isolates 3, 4, 5, 6, 1 and 9
significantly increased the nitrogen content of the soil after harvest. In addition, isolates
10, 3 and 9 gave the lowest percent clubrootinfection of 26.66 %, 40 % and 46.67%. The
same isolates gave the lowest club root severity infection of 2.3 and 2.67. In tems of
vegetative fresh weight, isolate 1 gave the highest.
From the results, some bacterial isolates and actinomycetes-like organism from
sunflower soil improved seed germination rate and plant height, enhanced nitrogen
uptake and reduced club root severity.
30
Conclusion
Some isolates showed good effect on the growth and development of chinese
cabbage and were able to reduced clubroot infection and severity. Isolates 2 and 8
enhanced the height of chinese cabbage. While isolates 1 and 8improved the vegetative
weight of chinese cabbage. Percentage (%) nitrogen content analysis also showed that
presence of isolate 1,2,3,4,5,6,9 in the soil enhanced the availability of nitrogen in the
soil.Indicative that such isolates are good biofertilizers candidates. On the other hand
Isolates 3, 9 and 10 reduced club root severity of chinese cabbage so they can be used to
manage clubroot.
Recomendations
Based on the results, the folowing are the recommendations:
1. Characterizethe bacterial isolates that has a good effect on the growth of c.
cabbage and isolates that lessened the club root severity.
2. Nitrogen analysis of sample plants must be conducted to support the result of
nitrogen analysis from soils.
3. Conduct a study to find out if combination of two or more different isolate having
different effect is applicable to attain better result.
4. Determine the link which creates antagonism of beneficial bacteria
andPlasmodiophorabrassicae.
5. Conduct field experiment utilizing the treatments.
31
LITERATURE CITED
ANTONIO, M. S.1986.fungicidal evaluation against alternaria leaf spot (alternaria
brassicae) in chinese cabbage. BS Thesis (Unpub.). Benguet State University, La
Trinidad, Benguet.Pp 5.
ARIFUZZAMAN, M., M. R. KHATUN, and H. RAHMAN.2010. African Journal of
Biotechnology
Vol.
9.
http://www.academicjournals.org/AJB/PDF/pdf2010/19Jul/Arifuzzaman%2
0et%20al.pdf
BANIASADI,
F.
2009.Agricultural
and
Biological
Sciences.
http://www.wadatabase.com/categories/Agricultural-and-Biological-
Sciences/
BURGE,
H.2008.
The
Environmental
Reporter.
http://www.emlab.com/s/sampling/env-report-06-2008.html
HIRSCH, A. M.2009.Brief History of the Discovery of Nitrogen-fixing Organisms.
http:/ www.mcdb.ucla.edu/Research/Hirsch/imagesb/HistoryDiscoveryN2fixingO
rganisms.pdf
IGARASHI, Y., S. MIURA, M. AZUMI, T. FURUMAI and R. YOSHIDA. 2006.
Studies on Plant-associated Actinomycetes and Their Secondary Metabolites.
http://www.pgrsa.org/2005_Proceedings/papers/034.pdf
JEFFREY, L. S. H. 2008. Isolation, Characterization and Identification of
Actinomycetesfrom
Agriculture
Soils
at
Semongok,
Sarawak.
http://ajol.info/index.php/ajb/article/viewFile/59415/47710
LINDGREN, D. T. 1992.Vegetable Garden Seed Storage and Germination
Requirements.http://www.seedman.com/veggerm.htm
NINGTHOUJAM, D.S., S. SANASAM, K. TAMREIHAO and S. NIMAICHAND.2009.
AfricanJournal of Microbiology Research Vol. 3.
http://www.academicjournals.org/ajmr/PDF/Pdf2009/Nov/Ningthoujam%20
et%20al.pdf
SHAHIDI BONJAR, G.H., P. R. FARROKHI, S. AGHIGHI, L. SHAHIDI BONJAR.
and
A.
AGHELIZADEH.2005.
Plant
Pathology
Journal.
http://docsdrive.com/pdfs/ansinet/ppj/2005/78-84.pdf
SIVAKUMAR, K. 2008. Actinomycetes.http://ocw.unu.edu/international-network-on-
water-environment-and-health/unu-inweh-course-1- mangroves/actinomycetes.pdf
32
SULASTRI, 2005.The Use of Beneficial Microorganisms in Agricultural Practices.
http://nature.berkeley.edu/BeahrsELP/Newsletter%20-
%20Summer%202009/Bacteria_in_agriculture_Sulastri.html
TITUS, A. and PEREIRA, G. N. 2005.The Role of Actinomycetes in Coffee Plantation
Ecology.http://www.ineedcoffee.com/05/actinomycetes/
WAKSMAN,
S.A.
1959.
The
Ascomycetes.
http://www.archive.org/stream/actinomycetes01waks/actino
mycetes01was_djvu.txt
WAKSMAN, S.A. and A.T. HENRICI.1943. The NomenclatureAndClassification Of
The Actinomycetes.http://jb.asm.org/cgi/reprint/46/4/337.pdf
33
APPENDICES
Appendix Table 1. Percentage germination as affected by the different isolates and the
different dilution rates taken after two weeks
TREATMENTS
REPLICATES
TOTAL
MEAN
R
R
R
1
2
3
control
100%
100%
80%
280%
93.33%
isolate 1
60%
60%
100%
220%
73.33%
isolate 2
100%
60%
40%
200%
66.67%
isolate 3
40%
40%
40%
120%
40.00%
isolate 4
100%
80%
60%
240%
80.00%
isolate 5
80%
80%
80%
240%
80.00%
isolate 6
80%
80%
100%
260%
86.67%
isolate 7
60%
80%
80%
220%
73.33%
isolate 8
20%
100%
60%
180%
60.00%
isolate 9
40%
40%
60%
140%
46.67%
isolate 10
20%
40%
20%
80%
26.66%
34
Appendix Table 2.Seedling height (in) as affected by the different isolates and the
different dilution rates measured after six weeks
TREATMENTS
REPLIICATIONS
TOTAL
MEAN
R
R
R
R
1
2
3
4
T0D0
2.53
2.23
2.69
2.58
10.03
2.51
T1D1
2.33
2.99
3.43
3.2
11.95
2.99
T1D2
4.3
3.7
3.73
4.3
16.03
4.01
T1D3
3.76
3.71
4.06
3.61
15.14
3.79
Sub-Total
10.39
10.4
11.22
11.11
43.12
10.79
T2D1
3.28
2.4
2.93
2.83
11.44
2.86
T2D2
3.1
3.18
3.16
3.49
12.93
3.23
T2D3
3
3.44
3.94
3.8
14.18
3.55
Sub-Total
9.38
9.02
10.03
10.12
38.55
9.64
T3D1
3.64
4.14
3.94
4.21
15.93
3.98
T3D2
4.19
4.04
4.46
4.35
17.04
4.26
T3D3
4.59
4.19
4.6
3.6
16.98
4.25
Sub-Total
12.42
12.37
13
12.16
49.95
12.49
T4D1
3.43
3.93
3.96
3.63
19.93
4.98
T4D2
4.08
3.8
3.76
3.29
14.93
3.73
T4D3
3.58
3.16
3.38
3.16
13.28
3.32
Sub-Total
11.09
10.89
11.1
10.08
48.14
12.03
T5D1
2.83
2.47
2.71
2.84
10.85
2.71
T5D2
3.04
3.3
3.25
3.51
13.1
3.28
T5D3
3.66
3.54
3.56
3.36
14.12
3.53
Sub-Total
9.53
9.31
9.52
9.71
38.07
9.52
T6D1
2.26
2.39
3.36
4.43
12.44
3.11
T6D2
4.33
3.88
3.97
3.86
16.04
4.01
T6D3
3.75
3.43
4.34
3.1
14.62
3.66
Sub-Total
10.34
9.7
11.67
11.39
43.1
10.78
T7D1
3.69
2.19
3.34
3.44
12.66
3.17
T7D2
3.44
3.55
3.96
3.91
14.86
3.72
T7D3
4.11
435
3.8
3.7
15.96
3.99
Sub-Total
11.24
440.74
11.1
11.05
43.48
10.88
T8D1
2.84
2.73
3.11
3.14
11.82
2.96
T8D2
3.77
3.97
3.2
3.18
14.12
3.53
T8D3
4.09
2.42
4.01
2.63
13.15
3.29
Sub-Total
10.7
9.12
10.32
8.95
39.09
9.78
T9D1
3.18
3.44
3.48
4.4
14.5
3.63
T9D2
3.96
4.66
4.66
4.41
17.69
4.42
T9D3
4.23
4.38
4.44
3.73
16.78
4.2
Sub-Total
11.37
12.48
12.58
12.54
48.97
12.25
T10D1
4.11
3.35
3.8
5.34
16.6
4.15
T10D2
4.76
5.26
5.39
4.61
20.02
5.01
T10D3
4.67
4.31
5.43
4.22
18.63
4.66
Sub-Total
13.54
12.92
14.62
14.17
55.25
13.82
35
SIMPLIFIED DATA
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
2.51
Isolate 1
2.99
4.01
3.79
3.60d
Isolate 2
2.86
3.23
3.55
3.21d
Isolate 3
3.98
4.26
4.25
4.16b
Isolate 4
4.98
3.73
3.32
4.01d
Isolate 5
2.71
3.28
3.53
3.17d
Isolate 6
3.11
4.01
3.66
3.59cd
Isolate 7
3.17
3.72
3.99
3.63d
Isolate 8
2.96
3.53
3.29
3.26d
Isolate 9
3.63
4.42
4.20
4.08bc
Isolate 10
4.15
5.01
4.66
4.61a
MEAN
3.454b
3.92a
3.824a
Means with the same letter are not significantly different at 5% level DMRT
ANALYSIS OF VARIANCE
Source of
DF
SS
MS
Fc
Probability
variance
Factor A
9
20.332
2.259
8.1971
0.0000
Factor B
2
9.808
4.904
17.7941
0.0000
AB
18
4.179
0.232
0.8424
Error
90
24.803
0.276
Total
119
59.122
Coefficient of Variation- 14.09 %
36
Appendix Table 3. Root length (in) as affected by the different isolates and the different
dilution rates measured after six weeks
Treatment/Dilution
REPLICATIONS
Total
Mean
R1
R2
R3
R4
T0D0
5.53
07.23
07.63
10.07
30.46
7.62
T1D1
7.43
09.77
10.05
12.40
39.65
9.91
T1D2
7.90
12.3
9.03
11.67
40.9
10.23
T1D3
8.03
09.9
9.70
06.10
33.73
8.43
Sub-Total
23.36
31.97
28.78
30.17
114.28
28.57
T2D1
10.43
9.27
12.7
8.2
40.6
10.15
T2D2
10.70
10.9
9.3
8.03
38.93
9.73
T2D3
11.77
9.97
13.7
8.1
43.54
10.89
Sub-Total
32.9
30.14
35.7
24.33
123.07
30.77
T3D1
8.30
8.03
9.17
7.6
15.9
8.28
T3D2
9.70
9.87
8.7
10
19.7
9.57
T3D3
11.67
9.3
8.33
9.9
21.57
9.8
Sub-Total
29.67
27.2
26.2
27.5
57.17
27.65
T4D1
7.5
13.6
12.17
9.83
43.1
10.78
T4D2
11.2
10.7
11.6
10.73
44.23
11.06
T4D3
9.93
12.6
8.37
13.03
43.93
10.98
Sub-Total
28.63
36.9
32.14
33.59
131.26
32.82
T5D1
11.13
11.87
10.53
10.63
44.16
11.04
T5D2
11.8
13.13
8.23
8.6
41.76
10.44
T5D3
10.63
13.57
11.87
10.3
46.37
11.59
Sub-Total
33.56
38.57
30.63
29.53
132.29
33.07
T6D1
9.77
11.07
11.33
10.53
42.7
10.68
T6D2
11.33
9.23
13.53
12.47
46.56
11.64
T6D3
10.63
15.47
10.77
10.33
47.2
11.8
Sub-Total
31.73
35.77
35.63
33.33
136.46
34.12
T7D1
8.77
8.57
11.4
10.1
38.84
9.71
T7D2
11.4
5.93
6.93
8.73
32.99
8.25
T7D3
11.63
14.53
7.77
8.83
42.76
10.69
Sub-Total
31.8
29.03
26.1
27.66
114.59
28.65
T8D1
10.37
8.3
14.57
10.2
43.44
10.86
T8D2
9.16
16.37
10.47
9.9
45.9
11.48
T8D3
7.54
9.23
10.63
13.53
40.93
10.23
Sub-Total
27.07
33.9
35.67
33.63
130.27
32.57
T9D1
11.63
8.3
12.93
10.27
43.13
10.78
T9D2
9.47
8.33
11.67
8.7
38.17
9.54
T9D3
7.57
8.43
8.03
17.8
41.83
10.46
Sub-Total
28.67
25.06
32.63
36.77
123.13
32.57
T10D1
10.23
10.83
11
12.1
44.16
11.04
T10D2
11.27
11.73
8.4
14.1
45.5
11.38
T10D3
11.67
10.33
13.6
9.87
45.47
11.37
Sub-Total
33.17
32.89
33
36.07
135.13
33.79
37
SIMPLIFIED DATA
TREATMENTS
DILUTION RATE
MEAN
104
105
106
Control
N/A
N/A
N/A
07.62
Isolate 1
09.91
10.23
08.43
09.52
Isolate 2
10.15
09.73
10.89
10.26
Isolate 3
08.28
09.57
09.80
09.22
Isolate 4
10.78
11.06
10.98
10.94
Isolate 5
11.04
10.44
11.59
11.02
Isolate 6
10.68
11.64
11.80
11.37
Isolate 7
09.71
08.25
10.69
09.55
Isolate 8
10.86
11.48
10.23
10.86
Isolate 9
10.78
11.38
10.46
10.87
Isolate 10
11.04
09.54
11.37
10.65
MEAN
10.323
10.332
10.624
ANALYSIS OF VARIANCE
SOURCE
DF
SS
MS
Fc
Tabulated F
OF
.05
.01
VARIANCE
Factor A
9
124.983
13.887
0.9269
1.98 2.61
Factor B
2
14.110
7.055
0.4709
AB
18
192.109
10.673
0.7124
Error
90
1348.413
14.982
Total
119
1679.615
Coefficient of Variation- 35.79 %
38
Appendix Table 4. Initial and final percentage nitrogen content of soil samples, taken
before the introduction of bacteria and after harvest
TREATMENTS
REPLICATIONS
TOTAL
MEAN
R1
R2
R3
Initial samples
0.23
0.25
0.22
0.70
0.23c
T0 - control
0.25
0.24
0.23
0.72
0.24bc
T1 – isolate 1
0.30
0.32
0.31
0.93
0.31a
T2 - isolate 2
0.30
0.31
0.30
0.91
0.30a
T3 – isolate 3
0.29
0.37
0.35
1.01
0.34a*
T4 – isolate 4
0.30
0.39
0.29
0.98
0.33a
T5 – isolate 5
0.30
0.37
0.31
0.98
0.33a
T6 – isolate 6
0.32
0.35
0.26
0.93
0.31a
T7 – isolate 7
0.31
0.32
0.24
0.87
0.29ab
T8 – isolate 8
0.30
0.31
0.23
0.84
0.28abc
T9 – isolate 9
0.29
0.30
0.32
0.91
0.30a
T10 – isolate 10
0.27
0.33
0.26
0.86
0.29ab
Grand Total
10.640
Grand mean
0.296
ANALYSIS OF VARIANCE
SOURCE OF DF
SS
MS
Fc
Tabulated F
VARIANCE
.05
.01
Treatments
11
0.034
0.003
3.000*
2.22
3.09
Error
24
0.028
0.001
Total
35
0.063
Coefficient of variation = 8.95
*= Significant at 5% level of significance
39
Appendix Table 5. Effect of the different Isolates on the final height (inches) of Chinese
cabbage taken after three months
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
6.5
6.4
8.0
20.90
6.97
T1 – isolate 1
7.1
8.5
8.0
23.60
7.87
T2 - isolate 2
8.5
8.6
7.5
24.60
8.20
T3 – isolate 3
6.8
6.6
7.5
20.90
6.97
T4 – isolate 4
0.0
6.5
7.5
14.0
4.67
T5 – isolate 5
6.5
6.6
8.3
21.40
7.13
T6 – isolate 6
8.9
8.5
0.0
17.40
5.80
T7 – isolate 7
7.6
6.7
8.6
22.90
7.63
T8 – isolate 8
6.0
11
9.0
26.00
8.67
T9 – isolate 9
7.0
6.5
8.7
22.20
7.40
T10 – isolate 10
6.5
7.9
7.0
21.40
7.13
TRANSFORMED DATA
TREATMENTS RIPLICATIONS
TOTAL
MEAN
R1
R2
R3
T0 - control
2.65
2.62
2.92
8.19
2.73
T1 – isolate 1
2.75
3.00
2.92
8.67
2.89
T2 - isolate 2
3
3.02
2.83
8.85
2.95
T3 – isolate 3
2.70
2.66
2.83
8.19
2.73
T4 – isolate 4
0.71
2.65
2.83
6.19
2.06
T5 – isolate 5
2.65
2.66
2.97
8.28
2.76
T6 – isolate 6
3.07
3.00
0.71
6.78
2.26
T7 – isolate 7
2.85
2.68
3.02
8.55
2.85
T8 – isolate 8
2.55
3.39
3.08
9.02
3.01
T9 – isolate 9
2.73
2.65
3.03
8.41
2.80
T10 – isolate 10 2.65
2.90
2.74
8.29
2.76
40
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
2.499
0.2499
0.78
0.6510
TRT
10
2.499
0.2499
0.78
0.6510
Error
32
7.092
0.32223
Corrected Total 32
9.591
Coefficient of Variation = 20.95280%
Appendix Table 6. Percentage incidence of club root as affected by the different isolates
taken at harvest
TREATMENTS
REPLICATIONS
Total
mean
R
R
R
1
2
3
T0 - control
100%
100%
80%
280%
93.33%
T1 – isolate 1
60%
60%
100%
220%
73.33%
T2 - isolate 2
100%
60%
40%
200%
66.67%
T3 – isolate 3
40%
40%
40%
120%
40%
T4 – isolate 4
100%
80%
60%
240%
80%
T5 – isolate 5
80%
80%
80%
240%
80%
T6 – isolate 6
80%
80%
100%
260%
86.67%
T7 – isolate 7
60%
80%
80%
220%
73.33%
T8 – isolate 8
20%
100%
60%
180%
60%
T9 – isolate 9
40%
40%
60%
140%
46.67%
T10 – isolate 10
20%
40%
20%
80%
26.66%
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
12921.21212
1292.12121
3.44
0.0075
TRT
10
12921.21212
1292.12121
3.44
0.0075
Error
32
8266.66667
375.75758
Corrected Total 32
21187.87879
Coefficient of Variation = 29.34346%
41
Appendix Table 7. Club root severity as affected by the different isolates, taken at harvest
TREATMENTS
REPLICATIONS
Total
Mean
R
R
R
1
2
3
T
a
0 - control
6
5
5
16
5.33
T
abc
1 – isolate 1
3
4
5
12
4
T
abc
2 - isolate 2
5
4
2
11
3.67
T
c
3 – isolate 3
3
3
2
8
2.67
T
ab
4 – isolate 4
6
4
4
14
4.67
T
ab
5 – isolate 5
5
5
4
14
4.67
T
ab
6 – isolate 6
4
4
6
14
4.67
T
abc
7 – isolate 7
3
4
4
11
3.67
T
bc
8 – isolate 8
2
4
3
9
3
T
c
9 – isolate 9
3
3
2
8
2.67
T
c
10 – isolate 10
2
3
2
7
2.33
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
30.061
3.0061
3.67
0.0052
TRT
10
30.061
3.0061
3.67
0.0052
Error
32
18.00
0.8182
Corrected Total 32
48.061
Coefficient of Variation = 24.07228%
42
Appendix Table 8. Fresh weight of vegetative parts (g) taken at harvest
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
50
13
19
82.0
27.33
T1 – isolate 1
82
123
95
300
100
T2 - isolate 2
35
88
60
183
61.00
T3 – isolate 3
50
36
87
173
57.67
T4 – isolate 4
0.0
21
30
51.0
17.00
T5 – isolate 5
31
19
48
98.0
32.67
T6 – isolate 6
81
69
0.0
150
50.00
T7 – isolate 7
59
45
57
161
53.67
T8 – isolate 8
30
135
56
221
73.67
T9 – isolate 9
53
16
83
152
50.67
T10 – isolate 10
40
47
47
134
44.67
43
TRANSFORMED DATA
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
7.11
3.67
4.42
15.2
5.07
T1 – isolate 1
9.08
11.11
9.77
29.96
9.99
T2 - isolate 2
5.96
9.41
7.78
23.15
7.72
T3 – isolate 3
7.11
6.04
9.35
22.5
7.5
T4 – isolate 4
0.71
4.64
5.52
10.87
3.62
T5 – isolate 5
5.61
4.42
6.96
16.99
5.66
T6 – isolate 6
9.03
8.34
0.71
18.08
6.03
T7 – isolate 7
7.71
6.75
7.58
22.04
7.35
T8 – isolate 8
5.52
11.64
7.52
24.68
8.23
T9 – isolate 9
7.31
4.06
9.14
20.51
6.84
T10 – isolate 10 6.36
6.89
6.89
20.14
6.71
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
86.4394
8.6439
3.69
0.1474
TRT
10
86.4394
8.6439
3.69
0.1474
Error
32
122.7707
5.1259
Corrected Total 32
199.2100
Coefficient of Variation = 33.33650%
44
Appendix Table 9. Dry weight of vegetative parts (g), obtained seven weeks after air
drying
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
3.41
3.06
1.89
8.36
2.79
T1 – isolate 1
5.01
4.72
5.91
15.64
5.21
T2 - isolate 2
2.18
4.37
2.24
8.79
2.93
T3 – isolate 3
2.70
1.26
3.89
7.85
2.62
T4 – isolate 4
0.00
0.90
1.36
2.26
0.75
T5 – isolate 5
1.30
1.16
2.11
4.57
1.52
T6 – isolate 6
3.51
2.44
0.00
5.95
1.98
T7 – isolate 7
3.17
3.08
3.37
9.62
3.21
T8 – isolate 8
2.01
4.68
2.78
9.47
3.16
T9 – isolate 9
2.34
0.96
3.06
6.36
2.12
T10 – isolate 10 1.99
0.58
2.13
4.70
1.57
TRANSFORMED DATA
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
1.98
1.89
1.55
5.42
1.81
T1 – isolate 1
2.35
2.28
2.53
7.16
2.38
T2 - isolate 2
1.64
2.21
1.66
5.51
1.84
T3 – isolate 3
1.79
1.33
2.10
5.22
1.74
T4 – isolate 4
0.71
1.18
1.86
3.75
1.25
T5 – isolate 5
1.34
1.29
1.62
4.25
1.42
T6 – isolate 6
2.00
1.72
0.71
4.43
1.48
T7 – isolate 7
1.92
1.89
1.97
5.78
1.93
T8 – isolate 8
1.58
2.28
1.81
5.67
1.89
T9 – isolate 9
1.69
1.21
1.89
4.79
1.60
T10 – isolate 10
1.58
1.04
1.62
4.24
1.41
45
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
3.0480
0.3048
2.23
0.0564
TRT
10
3.0480
0.3048
2.23
0.0564
Error
32
3.0103
0.1368
Corrected Total
32
6.0583
Coefficient of Variation = 21.71296%
Appendix Table 10. Fresh weight of roots (g) taken at harvest
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
4.82
0.64
2.86
8.32
2.77
T1 – isolate 1
1.01
0.96
1.92
3.89
1.30
T2 - isolate 2
2.25
1.98
3.66
7.89
2.63
T3 – isolate 3
0.40
0.58
1.01
1.99
0.66
T4 – isolate 4
0
0.57
1.97
2.54
0.85
T5 – isolate 5
3.15
0.25
0.89
4.29
1.43
T6 – isolate 6
1.64
0.59
0
2.23
0.74
T7 – isolate 7
0.62
0.59
2.01
3.22
1.07
T8 – isolate 8
0.43
4.07
1
5.5
1.83
T9 – isolate 9
0.48
0.35
1.25
2.08
0.69
T10 – isolate 10
1.12
0.67
0.45
2.24
0.75
TRANSFORMED DATA
TREATMENTS
REPLICATION
Total
mean
R1
R2
R3
T0 - control
2.31
1.07
1.83
5.21
1.74
T1 – isolate 1
1.23
1.21
1.56
4
1.33
T2 - isolate 2
1.66
1.57
2.04
5.27
1.76
T
3 – isolate 3
0.95
1.04
1.23
3.22
1.07
T4 – isolate 4
0.71
1.03
1.57
3.31
1.10
T5 – isolate 5
1.91
0.87
1.18
3.96
1.32
T6 – isolate 6
1.46
1.04
0.71
3.21
1.07
T7 – isolate 7
1.06
1.04
1.58
3.68
1.23
T8 – isolate 8
0.96
2.14
1.22
4.32
1.44
T9 – isolate 9
0.99
0.92
1.32
3.23
1.08
T10 – isolate 10
1.27
1.17
0.98
3.42
1.14
46
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
1.9323
0.19323
1.27
0.3048
TRT
10
1.9323
0.19323
1.27
0.3048
Error
32
3.3461
0.1521
Corrected Total 32
5.2784
Coefficient of Variation = 30.04843%
Appendix Table 11.Dry weight of Roots (g) recorded after seven weeks of air drying
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
0.98
0.43
1.58
3
1
T1 – isolate 1
0.74
0.45
0.82
2.01
0.67
T2 - isolate 2
0.97
0.90
1.09
2.96
0.99
T3 – isolate 3
0.28
0.36
0.33
0.97
0.32
T4 – isolate 4
0.00
0.31
0.29
0.6
0.2
T5 – isolate 5
0.46
0.12
0.55
1.13
0.38
T6 – isolate 6
0.59
0.51
0
1.1
0.37
T7 – isolate 7
0.32
0.42
0.84
1.58
0.53
T8 – isolate 8
0.35
1.46
0.43
2.24
0.75
T9 – isolate 9
0.11
0.24
0.55
0.9
0.3
T10 – isolate 10 0.19
0.32
0.24
0.75
0.25
47
TRANSFORMED DATA
TREATMENTS
REPLICATION
TOTAL
MEAN
R1
R2
R3
T0 - control
1.22
0.96
1.44
3.62
1.21
T
1 – isolate 1
1.11
0.97
1.15
3.23
1.08
T2 - isolate 2
1.21
1.18
1.26
3.65
1.22
T3 – isolate 3
0.88
0.93
0.91
2.72
0.91
T4 – isolate 4
0.71
0.9
0.89
2.5
0.83
T5 – isolate 5
0.98
0.79
1.02
2.79
0.93
T6 – isolate 6
1.04
1
0.71
2.75
0.92
T7 – isolate 7
0.91
0.96
1.16
3.03
1.01
T8 – isolate 8
0.92
1.4
0.96
3.28
1.09
T9 – isolate 9
0.78
0.86
1.02
2.66
0.89
T10 – isolate 10
0.69
0.91
0.86
2.46
0.82
ANALYSIS OF VARIANCE
SOURCE OF
DF
SS
MS
F
Pr> F
VARIANCE
value
MODEL
10
0.5909
0.05909
2.66
0.0269
TRT
10
0.5909
0.05909
2.66
0.0269
Error
32
0.4891
0.02223
Corrected Total 32
1.07999
Coefficient of Variation = 15.05225%
Document Outline
- Evaluation of Bacteria and Actinomycetes-likeOrganisms against Club Root (PlasmodiophoraBrassicae) in Chinese Cabbage(BrassicaPekinensis)
- BIBLIOGRAPHY
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
- MATERIALS AND METHOD
- RESULTS AND DISCUSSION
- SUMMARY, CONCLUSION AND RECOMMENDATIONS
- LITERATURE CITED
- APPENDICES