IMPACT OF CERCOSPORA APII ON TEAK NURSERY
AND ITS MANAGEMENT IN VIVO
* N.Murthy and S. Lokesh
*Department of Studies in Sericulture Science
Department of Studies in Biotechnology
Manasagangotri, University of Mysore
Mysore 570 006 Karnataka, INDIA
ABSTRACT
The aerial parts such as stem, leaves and vegetative parts of teak showed a
variety of fungi. Compared to the fruits, aerial parts of teak showed heavy colonies of
Cercospora apii. In this study, the pathogenicity of the fungus was tested in nursery
beds indicated the severity of leaf spot and leaf blight diseases. In order to control
nursery diseases, saplings in nursery were treated with fungicides like Bavistin,
Captan, Captafol, bio-agents like Trichoderma harzianum and Pseudomonas
fluorescens. Among fungicides Bavistin proved two fold better over control, in
managing the diseases.bio-agents Trichoderma harzianum and Pseudomonas
fluorescens also reduced the diseases to some extent. However, their effectiveness
remain inferior to that of Bavistin. The fruits of teak treated with Cercospora apii
resulted in delayed emergence in the nursery beds proved the aggressiveness of the
fungus. Among fertilizers provided, N.P.K amendment to the soil improved the field
standing of the saplings in the nursery bed.
Key words: Teak saplings, leaf spot, leaf blight, seedling decay, fungicides, bioagents.
INTRODUCTION
Forest provides multi-purpose benefits as sources for timber, fuel and fodder,
minor forests products and also adding to the aesthetic value and recreational needs of
man. In recent days considerable effort is taken to increase the productivity of existing
forests and to raise the plantations to meet the increasing demand of industrial wood
and high grade timber. Diseases, insect pests constitutes major biological
determinants of productivity.Catastropic losses may occur in the event of an out break
of a disease or an insect pest. Therefore, it is essential to know both the cause and
remedial measures of disease. In India as a part of a forestation programme
1
economically important species like Teak, Deodar and Sandal etc. are being raised in
plantation for which the seedling stock is raised in nurseries. Large scale plantation
programmes have necessitated a demand for plant stock for which more number of
nurseries is being established. In forest nurseries large quantities of seeds are
collected and used to raise seedlings. Almost all groups of fungi are found to be
associated with the seeds of forest tree species. Therefore, in forest nurseries problems
like poor germination of seeds/ fruits and seedling disease are commonly encountered.
Fungi are known to cause considerable damage to sprouting of seeds and seedlings.
Since, the success of plantations in afforestation programme is dependent on quality
of seedlings much importance should be given towards the prevention and control of
seedling diseases at the nursery stages. Teak (Tectona grandis Linn.f) is one of the
most extensively planted tree species in afforestation programme because of its fast
growing nature fire resistibility, non browsability and high timber value. In Teak
nurseries fungal disease problems are known to raise commonly, which seriously
affect the planting stock and disturb the planting programme. Therefore, in order to
fill these lacunae and acquire a feasible method of controlling the nursery diseases,
present programme has been proposed to evaluate the aggressiveness of the fungus
Cercospora apii in nursery.
MATERIALS AND METHODS
Evaluation of fruits, seeds and vegetative cuttings of teak for the occurrence of
mycoflora
Matured fruits of Teak collected from forest nursery situated in Nagavala, Mysore
district of Karnataka State and were stored in cloth lined bags at room temperature
conditions ( 28±20 C ) for further studies. Seeds were obtained from the teak fruits
manually by crushing the fruits wall with the care taken not to damage the seeds. In
forest nursery collapsing of saplings was observed in large number at different growth
stages due to different casual organisms may be from air, soil water or from plant
materials itself. Based on these observations for the purpose of identifying the disease
causing organisms, vegetative parts like, petiole, and tender branches of nursery
saplings were collected and maintained at 50 C for further studies. Each of 400 fruits
and seeds of the collected samples were plated on three layers of wet blotters
equidistantly in perspex plates and were incubated for a period of one week under
2
12/12 hour alternate cycles of light (Near Ultra Violet Light) and darkness at 22±20 C.
Similarly, vegetative cuttings of diseased plant were washed in water for 4 - 5 times,
air dried and cut into small pieces of 1 cm length. 400 bits of vegetative parts were
randomly picked, plated on wet blotters and incubated according to the standard
conditions of temperature and humidity. On 8th day of incubation the fruits/seeds as
well as the vegetative bits were evaluated thoroughly with the aid of stereo-binocular
and compound microscope for the occurrence of their associated mycoflora. In case of
fruits and seeds, the germination was noticed on 10th day of incubation. In all the
cases, the results were recorded and tabulated.
Effect of spore suspension and culture filtrate of Cercospora apii on fruits/seeds
germination and seedling growth
The dominant fungus Cercospora apii isolated from infected plant parts
maintained and multiplied on Potato Dextrose Agar (PDA) medium in Petri plates.
The spore suspension as prepared from 8th day- old sporulated colonies, using distilled
water. The spore load was assessed haemocytometrically (5x 106 spores/ml) and the
same was mixed with the paste of mycelial mat. On the other hand, the culture of the
fungus was grown of Potato Dextrose Broth (PDB) medium in a series of 250 ml
conical flasks . On 12th day of incubation the culture filtrate was collected and filtered
through Whatman No.1 filter paper and was stored at 5o C for further use. The treated
fruits were then sown in soil beds according to normal procedures and practices. The
soil beds were irrigated regularly to avoid desiccation. On 20th day of sowing, the
seed beds were observed for the occurrence of emerging seedlings. At every intervals
of 2 days from the 1st observation, the seedlings emerged were counted and the
growing on observations were made with respect to the expression of any type of
symptoms.
Effect of treating agents on the incidence of nursery diseases of teak
Forest nursery maintained in Nagavala Forest Nursery Research Station (FNRS),
Mysore was considered for the routine observations with special reference to nursery
diseases of teak. For this purpose 6 month old saplings raised out of fruits were
selected in the nursery beds. Initially in the nursery there were many kinds of diseased
plants with symptoms varied from leaf spots of smaller and large size, leaf necrosis,
3
leaf blight and wilt. Healthy plants showed no such symptoms were interspersed
along with the diseased ones. In order to find out an efficient treating agent, in the
present of study, different chemical fungicides like Bavistin, Captan, Captafol, bioagents like Trichoderma harzianum and Psuedomonas fluorescens were used to spray
over the diseased nursery blocks of equal dimension measuring 2mx2m were
identified and randomly selected for foliar spray. The selected fungicide solutions
were separately prepared at 0.2 % concentration in water and were sprayed on to the
plants of identified blocks. Similarly, the biological agents like Trichoderma
harzianum and Psuedomonas fluorescens were used to spray over the plants of
selected blocks in the nursery. In this case, the spore suspension of Trichoderma
harzianum was obtained from 8th day- old sporulated colonies. The spore load was
calculated to be 5x105 spores /ml using haemocytometer. In case of Psuedomonas
fluorescens two day- old culture in King’s B broth was used. On 0th day itself the total
numbers of plants / block were recorded. Apart from this diseased plants showing leaf
spots, leaf necrosis, leaf blight, wilt were recorded and similar observations were
made at every interval of 10 days after treatment. The variations in the disease
incidence that developed after spray treatment were recorded and tabulated. A long
with these observations other parameters like top growth of the plants, total number of
leaves / plant were recorded based on the average of 25 plants separately for both
healthy and diseased ones.
Response of fertilizers on the incidence of seedling diseases of teak due to
Cercospora apii
Soil ingredients are also known to affect the plant growth. In presence of
other biotic factors, nitrogen rich fertilizers are known to stimulate the activities of
pathogenic species in the host system result in the succumbance of the host to the
fungal diseases. Depending upon the nature of soil and age of the plant, the
application of fertilizers will be varied. Keeping this view in mind in the present
study, different synthetic fertilizers like Urea, D.A.P and N.P.K ( 17:17:17) were
applied to the 6 month old nursery beds at the rate of 25 gm/m2 area. On the same day
the saplings were sprayed with the culture suspension of the Cercospora apii. Then
the plant growth and disease systems were recorded at equal intervals of 10 days.
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RESULTS AND DISCUSSION
Varied data with respect to fruits, seeds and vegetative parts of teak
represented in Table - 1 indicates a variety of fungal species. The degree of fungal
incidence was highest in fruits compared to seeds. Fruits and the aerial parts showed
higher incidence of fungi. Among fungi Aspergillus niger, Chaetomium globosum,
Cunninghamella sp., Fusarium moniliforme, Rhizopus stolonifer, Sphaeronema sp.,
Actinomycete species were recorded above 50%. Other fungi like Fusarium
semitectum and Fusarium solani were also recorded at higher incidence. In case of
seeds, since it is deep seated and having good protective wall, devoid of many fungal
species. However, some Aspergilli like Aspergillus niger, Aspergillus versicolor,
Aspergillus flavus, Chaetomium globosum, Rhizopus stolonifer, Actinomycte species
were recorded at moderate level. The incidence of fungi in the vegetative parts was
quite high due to continuous exposure through out its growth. The fungi recorded in
vegetative parts were Aspergillus niger Aspergillus versicolor, Aspergillus flavus,
Chaetomium globosum, Cunninghamella sp, Actinomycte sp., Fusarium moniliforme
Fusarium semitectum, Actinomycte species and Cercospora apii (Table-1).
Table-1: Assessment of fungi associated with the fruits, seeds and vegetative parts of
teak
Mycoflora
% incidence of fungi in different components
Vegetative parts
Fruits
Seeds
-
6
26
A. niger
26
9
23
A. versicolor
43
10
25
Actinomycete sp.
65
6
15
Cercospora apii
-
-
30
Chaetomium globosum
86
5
27
Cunninghamella sp
76
-
27
Fusarium moniliforme
36
-
13
F. solani
Aspergillus flavus
(young shoot)
13
-
-
F. semitectum
-
-
27
Macrophomina phaseolina
-
-
23
Myrothecium roridum
-
-
29
Mucor sp.
33
-
-
Rhizopus stolonifer
50
25
-
Sphaeronema sp.
60
-
-
Aspergillus ochraceus
26
-
-
% seed germinations
20
10
-
*Data based on 400 Fruits/Seed /units
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Interestingly, the incidence of Cercospora apii was 30% in the aerial parts. There was
no such incidence observed in fruits and seeds. This indicated that the Cercospora
apii lodged over the vegetative parts of the plants at higher incidence and made the
plants susceptible to the leaf spot and blight diseases. The aggressive nature of
pathogenic fungi results in the depletion of seed germination or otherwise results in
the pre/post emergence mortality of the seedlings. The data provided in Table-2
indicated the pathogenicity of Cercospora apii on seed germination of teak.
Irrespective of the fungal species as well as their culture filtrates there was depletion
in the rate of field emergence over control.
Table-2: Effect of Cercospora apii on fruit/seed germination of teak
Days after sowing
%f fruit/seed emergence in presence of Cercospora apii and its culture filtrate
Control
Spore suspension
Culture filtrate
20
1
-
-
22
3
-
-
24
6
-
-
26
8
1
1
28
9
1
2
30
11
2
4
Control = not treated with fungal suspension /culture filtrate
Normally, in teak, germination starts from 20th day of incubation (sowing).
Even on 30th day of incubation, there was limited number of emergence of seedlings
compared to control. Among fungi, Cercospora apii was found to be highly
aggressive which resulted in poor germination (Table-2). The efficacy of different
fungicides against the potency of leaf spot causing species Cercospora apii has been
tested and the results were tabulated in Table-3. The symptoms such as leaf spot and
leaf blight were decreased in all the treatments, over control. Among fungicides,
Bavistin provided its efficacy in reducing the disease even on 30th day of plant
growth. The bio-agents, Trichoderma harzianum and Psuedomonas fluorescens also
played an important role in diminishing the disease to some extent. In all the
treatments, there was gradual recovery of plants from disease. Compared to bioagents, fungicides controlled the disease to a greater extent. The application of
treating agents improved the growth of the plants to a higher extent over control. In
case of Bavistin treatment the disease recovery was assessed upto 25% over its
corresponding control. It was observed that the Captafol was found to be the least
6
effective in reducing the disease whereas the Captan stood medium in its
effectiveness. Though the bio-agents gave better results over control remained inferior
to that of fungicides. There was no much difference among bio-agents used against
the disease causing organisms.
The saplings treated with culture filtrate of Cercospora apii were found to
have some detrimental effect. In this case also both fungicides and bio-agents
improved the plant growth over control. Symptoms like leaf spot and blight were
gradually minimized as the plant ages. Bavistin gave promising results in reducing the
disease over control
by 20%. Captan stood medium in its effects against disease
causing organisms. But, Captan and Psuedomonas fluorescens failed to give better
results, comparatively (Table-3). Among the fertilizers used, the plants with N.P.K
(17:17:17) showed less incidence of disease. In case of plants provided with urea and
D.A.P no much variation were observed with respect to plant height, girth, number of
leaves, leaf spot and leaf blight diseases. In both the cases of Cercospora apii fungal
suspension and culture filtrate, similarities were observed with respect to growth
parameters and disease symptoms irrespective of fertilizers amended to the soil. It
indicates the uptake of nutrients will be in trace amount, which might, be the reason
for indifference observed among the different treatments. Usually fertilizers enriched
with nitrogen are known to increase the expression of disease symptoms due to rapid
establishment of the fungus in the host systems (Table-4). A variety of plant species
grown in a common place tend to spread the diseases. The disease causing organism
may invade the plant through root, leaves young axillary buds or any other parts of the
plants where there is mechanical damage. The building up of inoculum in particular
location results in the easy spreading of the same on to the surrounding plants. In the
present investigation, leaf and stem parts of teak having spots, blight and wilt
indicated an array of fungi. The association of Fusarium, Myrothecium and some
Aspergilli might have played a major role in the expression of disease symptoms. In
the present findings, fruits showed heavy incidence of fungi compared to seeds and
vegetative parts. Fruits provided sufficient shelter and nutrients to a variety of fungi.
Different enriched flora in the fruits may be due to contamination with fungi through
soil. Some fungi like Chaetomium, Humicola and Cytaladium species are reported to
be cellulolitic in nature. Cellulolitic species are known to results in weathering of teak
fruits.
7
Table-3: Effect of different treatments on the incidence of some nursery diseases of teak
Treatments
Variation in the disease symptoms due to Cercospora apii and its culture filtrate
Leaf spot %
Leaf blight %
Mean %
disease
Seedling decay %
incidence
A
B
C
D
40
25
15
13
(35)
( 18)
(16)
( 14)
45
40
39
A
B
14
11
C
D
A
B
C
25
20
D
Fungicides
Bavistin
Captan
(33) ( 30) (29)
Captafol
43
42
40
(31) ( 22) ( 17)
Bio-agents
42
Trichoderma
(33) (33)
40
36
( 26)
39
(22)
37
34
(21)
( 28)
43
39
37
(39)
( 25)
(29)
( 28)
50
50
48
46
8
(12) ( 11) ( 9)
14
13
12
(22) (24) ( 20
16
15
14
7
18
16
14
13
( 8)
(32) ( 24) ( 16) ( 10)
10
33
( 19)
14
(24) ( 17) (19) ( 9)
(16) (14)
34
12
(13) ( 17)
32
39
(35) ( 30) ( 29)
36
33
34
30
25
(21d)
27
33b
( 26)
(30c)
29
29b
(39) (40) ( 37 ) (35)
35
24c
(38a)
25
29b
(26)
(30c)
25
31b
(33)
(35ab)
28
31
30b
(30) (33) (32 )
(31)
(32bc)
38
40a
(33)
(35ab)
(37) (28) (27 )
harzianum
44
Pseudomonas
26
23
21
19
35
30
34
(18) (16) ( 13) ( 14)
(37 (37) (34)
25
30
fluorescens
Control-1
Control -2
(39)
( 39 ) (47)
50
48
(47)
46
30
(42 ) ( 71) ( 61)
( 60)
20
17
(15) ( 18) (18)
29
28
28
(16) (20) (30)
A = 0th day observation
Control 1= without fungus
B = 10th day observation
Control 2 = With fungus
16
(16)
28
( 30)
40
30
40
39
(38) ( 36) ( 34)
th
C = 20 day observation
D = 30th day observation
*Data given in the parenthesis represents disease symptoms due to culture filtrate of Cercospera apii
* Data indicated by different superscripts differ significantly at P = 0.05 level (DMRT)
8
Table-4: Effect of different synthetic fertilizers on the incidence of some nursery diseases of teak
Treatment
Variation in the disease symptoms due to Cercospora apii and its culture filtrate
Leaf spot %
A
Leaf blight %
B
C
42
15
13
(36) ( 29)
(30)
( 28)
39
35
17
15
(30)
(29) (35)
N.P.K
41
21
11
(17:17:17)
( 24
(20)
(19)
(17)
Control-1
45
42
40
39
Urea
D.A.P
40
(40)
Control -2
A = 0th day observation
th
B = 10 day observation
( 39 ) (35)
D
( 31)
9
(32)
50
50
41
40
(47 )
( 42)
( 46)
( 42)
A
B
20
20
disease
Seedling decay %
C
12
Mean %
D
11
A
28
B
26
C
25
D
24
(20) ( 19) ( 18) (22)
(31) ( 29) ( 29) ( 27)
20
29
17
13
(29) (40) ( 18)
12
14
10
(23) ( 14) (12
29
( 19)
9
( 11)
16
15
(19) ( 16) (14)
(12)
26
16
12
17
16
(24 ( 20) (16)
Control 1= Without fungus
Control 2 = With fungus
16
( 15)
26bc
(29ab)
25c
25
18
(29) ( 29) ( 27
( 25)
(28b)
13
21d
(30 ) (27) ( 24)
(22)
(26b)
29
27
29ab
29
28
incidence
25
29
16
28
(29) (30) (29 )
31
31
29
(31) ( 31) ( 32)
(30)
(30ab)
27
30a
(32)
(32a)
DAP = Di-Ammonium Phosphate
NPK= Nitrogen, Phosphorous, Potassium
C = 20th day observation
D = 30th day observation
*Data given in the parenthesis represents disease symptoms due to culture filtrate of Cercospera apii
* Data indicated by different superscripts differ significantly at P = 0.05 level (DMRT)
The increase in germination of fruits is most probably due to the preweathering of fruits which has been facilitated the seeds for the easy sprouting. The
present findings are in confirmatory with the observations of Dadwal and Jamaluddin
(1988). Fruits treated with Cercospora apii and its culture filtrate resulted in the
diminished germination compared to their corresponding control. This indicated the
toxigenicity of the fungus to the fruits. The pathogenicity of the fungus is indexed by
the delay in germination, which is in correlation with the field emergence. The
production of toxins by Fusarium solani hindered the development of Eucalyptus
plants was reported by Kumar and Viswanath (1993) remained parallel with the
present findings. There are several reports indicated the practice of foliar spray in the
nursery to get rid of fungal diseases in the field (Mehrotra, 1998; Segal et al., 1989;
Sujana Singh and Pandey, 1989; Dadwal and Jamaluddin, 1996 and Murthy et al.,
2004). The fungicide, Bavistin gave better result over other fungicides used. These
fungicides might have interacted with the metabolites produced by the pathogen in the
9
plant system. Hence, the toxic effect of the pathogen might have diminished
successfully or otherwise the Bavistin compounds might have resulted in the
formation of chelates or some other complex compounds along with the host
ingradients which might have resulted in the failure of proper, sufficient nutrient
supply to the pathogen. The other alternative way might be due to the induction of
some defensive factors. In the host it might have induced in response to chemical
application. The effectiveness of Bavistin in reducing the disease is also evident by
the findings of Siddaramaiah et al. (1980) who have assessed the effect of the same in
controlling the leaf spot disease of Wrightia tinctoria. The inefficacy of Captan might
be due to the lower dosage that has been preferred for spraying. It also possible that
the chemicals are often host specific in nature. Apart from these, texture, type and
nature of the host also play an important role in the retention of chemicals. There are
some reports available indicated Dithane M-45 is superior over Bavistin in controlling
the Anthracnose of tree species. Some of these informations gives an idea about the
necessity of screening of an array of fungicides to control the leaf spot and leaf blight
in forest tree species. In nature microbes’ exhibits antibiosis, hyphal parasitism
through some molecular weapons like enzyme, toxins and phenolics etc. In fungi
hyperparasitism, lysis formation, coiling are common phenomenon results in the
inhibition of the weaker species. Based on this antagonistic activity, fungi have been
exploited for controlling a variety of pathogenic species. In the present findings, the
usage of Trichoderma harzianum and Psuedomonas fluorescens benefited to some
extent in controlling diseases. The variation among the bio-agents in controlling the
disease may be due to the aggressive nature and also depending upon the production,
type nature and quantity of secondary metabolites.
The inhibiting activity of some pathogens in forest tree species through some
biological agents has been discussed by many workers to find an alternative way
against the usage of fungicides. The data obtained in the present findings indicated an
idea for screening an array of fungi for their antagonistic ability to protect the plant
disease causing fungi. Pathogen interferes with upward or downward movement of
inorganic nutrients and water, which results in severity of disease in plants. The
response of fertilizers and micronutrients in relation to plant development in nursery
has been discussed by Chinnathuri et al. (1997). The variability in the expression of
diseases also varies with respect to change in pH, binding property of the soil due to
10
amendment of synthetic fertilizers. Improvement in plant growth inspite of fungal
infection might be due to strengthening of root system through the adequate supply of
fertilizers in the initial days of plant development.
ACKNOWLEDGEMENT
The authors wish to express sincere thanks to University Grant Commission
for providing the necessary funds. We wish to thank University of Mysore and
Department of Studies in Sericulture Science, Manasagangothri, University of
Mysore, Mysore for extending the laboratory facilities.
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