Download PDF - Research Journal of Agricultural Sciences (RJAS)

Antagonistic Effect of Fungi Associated with Apple Scab Lesions on
Growth of its Pathogen Venturia inaequalis (Cke.) Wint.
I M Sharma
Horticultural Research Station,
Dr. Y. S. Parmar University of Horticulture and Forestry, Seobagh, P.O. Neoli, District Kullu-175 138, H.P., India
e-mail: imsharmakulu@hotmail.com
A B S T R A C T
Amongst three fungi viz Trichothecium roseum, Penicillium expansum and Alternaria mali identified
occurring in association with scab lesions caused by Venturia inaequalis on apple fruits; T. roseum was
prevalent in highest frequencies (73-81%). From these three fungi evaluated for their antagonistic effect
against Venturia inaequalis, T. roseum alone coiled and penetrated the mycelium of scab fungus. In vitro
studies also indicated that T. roseum drastically altered its morphology and conidial production. T. roseum
also resulted in maximum inhibition in the production (99.6%) and germination (72.1%) of conidia of V.
inaequalis besides delaying its formation by 35 days as shown by dual culture studies. P. expansum
followed by A. mali were less effective. Furthermore, T. roseum also registered maximum inhibition in
development of pseudothecia (44.7-100%) and subsequent ascospores productivity (30.9-100%) on fallen
leaves at its inoculum density ranging between 0.2x10 4 -1.5x10 4 cfu/ ml. The next best fungus was P.
expansum, which inhibited the pseudothecia formation and ascospore productivity between 18.1-52.2
and 12.6-56.4 per cent, respectively, whereas, A. mali was less effective.
Key words: Alternaria mali, Apple, Penicillium expansum, Scab, Trichothecium roseum, Venturia inaequalis
Amongst various diseases affecting apple, scab
caused by Venturia inaequalis is the most destructive
and caused epiphytotics in early eighties and mid
nineties (Gupta 1978, Gupta et al. 1984, Sharma et al.
1995). Presently, this disease is managed effectively by
giving 7-8 consecutive sprays with different fungicides
during the crop season (Anonymous 2003). These
treatments are highly expensive and render the fruit
covered with chemical residues that could be harmful.
Moreover, adoption of this extensive spray programme
has also resulted in the development of fungicide
resistance strains of this pathogen, particularly against
carbendazim and dodine fungicides (Basu and Puttoo
1984, Gupta and Sharma 1996, Gupta et al. 2000). A
practical approach in controlling this disease involves
the disruption of disease cycle by not allowing the
development of its saprophytic (perfect) stage i e
formation of pseudothecia, which develop on fallen
infected leaves during winter months (December-
February). These pseudothecia mature in the first week
of March to release ascospores, a primary source of
inoculum, to cause infection in spring (Gupta and Raj
1988, Sharma and Gupta 1996). The use of a urea spray
at very high concentration (5%) at pre-leaf fall stage
(first fortnight of November) is recommended for an
early fall of leaves and their quick decomposition. Urea
treated leaves do not support the formation of
pseudothecia and thereby interrupt the disease cycle
(Gupta and Lele 1980a). However, application of urea
at such a higher concentration caused injuries and
predisposed the plants to the attack by canker pathogens
(Thakur and Sharma 1999). Like urea, use of various
Research Journal of Agricultural Sciences 2010, 1(3): 245-248
245
microbial antagonists, isolated from fallen leaves
resulting in quick decomposition of leaves, have also
been successfully used to break the disease cycle of
target pathogen (Andrews et al. 1983). Since, various
fungi have been observed to occur in association with
scab lesions on apple fruits under field and storage
conditions, efforts were made to isolate, identify and
evaluate these fungi for their efficacy on growth
inhibition, hyper-parasitism, conidia formation and
germination of V. inaequalis under in vitro conditions
for two successive years 2004-05. In addition to these in
vitro studies, in vivo studies were carried out on
development of V. inaequalis pseudothecia on fallen
infected leaves and subsequent ascospores productivity
over three consecutive years 2005-2008.
MATERIALS AND METHODS
Isolation, identification and incidence of different fungi
occurring in association with scab lesions on apple
fruits
Three hundred scab-infected fruits were collected
from each of four different locations (Jangelli, Shalang,
Nagujohar and Blarga/ Tonk) during the months of
June–August during the year 2004 and 2005 in
Himachal Pradesh, India. Various fungi occurring in
association with scab lesions were observed under
microscope and identified (Barnett 1969). Per cent
incidence of their occurrence with scab lesions was
recorded. The fungi occurring in higher incidence were
isolated on potato dextrose Agar (PDA) medium and
maintained for further studies.

Studies on antagonistic effect of test fungi on growth,
morphology and, production and germination of
conidia of scab pathogen (Dual culture method)
The test fungi isolated as mentioned above were
screened for their antagonistic activities by dual culture
technique (Huang and Hoes 1976). Since scab fungus is
an extremely slow growing in vitro, and therefore the
culture discs of these test fungi (2mm) taken from the
margins of the vigorously growing seven days old
culture were aseptically transferred to solidified PDA
on opposite side (5.0cm apart) of 20 days old culture
(12mm) of scab fungus in Petri plate (9cm). The Petri
plates containing the culture bit of pathogen alone were
kept as control. These plates were then incubated at
20±1°C for 8 days and per cent growth inhibition (PGI)
was calculated by adopting the procedure suggested by
Vincent (1947). PGI = C-TC×100, where C = mycelial
growth (mm) in control and T = mycelial growth (mm)
in treatment. The nature of antagonistic action was also
recorded by observing (inhibition zone) under
microscope. Further, the effect of these test fungi on
morphology of mycelium and conidia of V. inaequalis
were also recorded by observing them under
microscope. These dual culture plates were also
periodically observed for formation of conidia of V.
inaequalis in order to record the delay in conidial
formation. The effect of these test fungi on conidial
production of V. inaequalis was studied by placing a
2mm bit of fungal mycelium aseptically placed on
opposite side of the culture (5cm apart) from a 25 days
old culture (16 mm) of V. inaequalis and comparing
amount of conidia produced in control as well in dual
culture plates were counted after 10 days of incubation
at 20±1°C with the help of haemocytometer. Per cent
inhibition in conidial production (PICP) was calculated
by adopting the procedure suggested by Vincent (1947).
The effect of these test fungi on germination of conidia
of V. inaequalis was studied in another experiment by
putting two drops of its conidial suspension containing
fixed number of conidia (500/ ml) harvested from dual
culture plate into a cavity slide. These cavity slides
were then kept in Petri plate fitted from inside with
moist filter paper and incubated at 221 o C for two days.
Numbers of spores germinated were counted under
microscope for each test fungus (10 microscopic fields)
and per cent inhibition in germination was calculated by
using the formula as described above.
Table 1 Effect of different fungi occurring in association with scab lesions on apple fruits on morphology
(mycelium and conidia), conidia production and their germination of scab fungus, V. inaequalis
Parameter # V. inaequalis T. roseum and V. P. expansum and V. A. mali and V. inaequalis
inaequalis
inaequalis
Mycelium Brown to mousy black 3.92-
6.84m in width
Brown to mousy black
3.92-5.89m in width
Brown to grey black 3.92-
6.24m in width
Brown to grey black 3.92-
6.32m in width
Conidia
0-1 septate, obpyriform to
obclavate (flame shaped), pale
to mild olivaceous ranging
between 11.62-31.32×5.86-
7.22m (Av. 22.42×6.65m)
Single celled, pyriform to
clavate, pale to olivaceous
ranging between 11.62-
22.36×5.86×9.18m (Av.
16.52×7.64m)
Single celled, obpyriform
to clavate shaped, pale to
mild olivaceous, ranging
between 11.62-
29.31×5.86-7.52m (Av.
21.92×6.82m)
Single celled, obpyriform
to clavate, pale to mild
olivaceous, ranging
between 11.62-
30.62×5.81-7.34m (Av.
22.16×6.74m)
Delay in conidia
--- 35 21 16
formation (days)
Conidial
9.2×10 4 3.5×10 2 4.8×10 3 7.4×10 3
production/ ml #
Conidial
85.4 23.8 (72.1)* 55.3 (35.2)* 64.3 (24.7)*
germination (%)
#Average of 10 observations *Per cent inhibition in spore germination
Effect of test fungi on development of pseudothecia and
ascospores productivity of apple scab pathogen
Scab infected leaves were collected from the hot
spots (apple orchards of Janjelli, Shalang, Nagujohar,
Blarga, and Tonk in Himachal Pradesh, India) of scab
disease in the first week on November during each of
the three consecutive years 2005-06, 2006-07 and 2007-
08. Eight inoculum levels (cfu/ ml) of each test fungus,
occurring in association with scab lesions, were
prepared by serial dilution method (Table 2). Triton, a
sticker, was added in each at a concentration of 0.02 per
cent. Forty scab infected leaves were separately dipped
in the earlier prepared concentration of these fungi for 5
minutes. These were then put in a nylon bags and kept
on orchard’s floor at Shalang (one of the hot spot of
scab), in Kullu district till spring season of the
following year. In control treatment, scab infected
I M Sharma
246
leaves were dipped in sterilized water containing Triton
(0.02%). The leaves were examined for formation of
pseudothecia and ascospore productivity by adopting
the procedures described by Gupta and Lele (1980a)
and Gupta (1981), respectively.
Statistical analysis
Data recorded for different years were pooled for
each test fungus and were subjected to statistical
analysis by following the method of variance described
by Gomez and Gomez (1984) to find out the least
significant difference (LSD) amongst the treatments at 5
per cent level.
RESULTS AND DISCUSSION
Identification of fungi occurring in association with
scab lesion on apple fruits and their relative incidence

Antagonistic Effect of Fungi Associated with Apple Scab
In all four fungi namely; Trichothecium roseum,
Penicillium expansum and Alternaria mali and
Glomerella cingulata were identified occurring in
association with scab lesions of apple fruits. Amongst
these the T. roseum was most prevalent and its
incidence varied between 73-81 per cent. It was
followed by A. mali (10-13%), P. expansum (8-11%)
and G. cingulata (1-3%), respectively. Kaul (1985),
Gupta and Verma (1986), Sharma and Bhardwaj (2000)
and Sharma (2004) also observed these fungi occurring
in association with scab lesions and later these cause
rots in apple fruits during storage.
Table 2 Effect of three fungi associated with scab lesions on pseudothecia formation and ascospore productivity
Inhibition (%) in ascospore productivity/ ml by test fungi *
Inoculum
Reduction (%) in pseudothecia number/ cm 2 on fallen
density
leaves by test fungi *
(cfu/ ml×10 4 ) T. roseum P. expansum A. mali. T. roseum P. expansum A. mali.
2.0 100.0 (90.0)# 52.2 (46.26) 41.2 (39.33) 100.0 (90.0) 56.4 (48.68) 44.8 (42.02)
1.5 100.0 (90.0) 51.1 (45.63) 40.1 (39.29) 100.0 (90.0) 54.2 (47.41) 42.5 (40.69)
1.2 94.8 (76.82) 49.4 (44.66) 36.4 (31.11) 100.0 (90.0) 50.4 (45.25) 40.2 (39.35)
1.0 89.1 (70.72) 45.5 (42.42) 33.8 (35.55) 98.5 (82.97) 45.4 (42.36) 37.7 (37.88)
0.8 82.4 (65.20) 40.2 (39.35) 30.1 (33.27) 93.1 (74.77) 36.8 (37.35) 31.3 (34.02)
0.6 74.9 (59.93) 34.9 (36.21) 27.3 (31.50) 82.5 (65.27) 28.3 (32.77) 24.1 (29.40)
0.4 62.3 (52.12) 27.2 (31.44) 20.3 (26.78) 60.5 (51.06) 22.5 (28.32) 17.4 (24.65)
0.2 44.7 (41.96) 18.1 (25.18) 11.8 (20.09) 30.9 (33.77) 12.6 (20.79) 8.5 (16.95)
Control (Water) 181.2 * * 124.4x10 3$
LSD (P=0.05)
LSD (P=0.05)
Due to fungi = 2.62 Conc. = 1.96 Fungi x Conc. = 3.09
Due to fungi = 2.54 Conc. = 1.42 Fungi×Conc. = 3.72
*Average of three years (2005-06, 2006-07, 2007-08) observations, #Figures in parenthesis are the angular transformed values,
**No. of pseudothecia/ cm 2 of leaf, $Ascospore productivity/ ml
Antagonistic effect of test fungi on growth, morphology,
conidia production and their germination of V.
inaequalis (Dual culture studies)
In vitro evaluation of former three fungi by dual
culture technique indicated that colony of test fungi
overgrew the colony of scab fungus V. inaequalis, as it
grows at very slow speed in the medium. Further,
microscopic studies revealed only T. roseum resulted in
hyper- parasitism action. The hyphae of T. roseum
coiled and penetrated the hyphae of V. inaequalis
whereas; the hyphae of other fungi neither coiled nor
penetrated the mycelium of V. inaequalis. The studies
on the effect of these fungi on morphology of mycelium
V. inaequalis indicated that T. roseum resulted in drastic
decrease in hyphal width of V. inaequalis without
affecting its color, whereas other two fungi altered the
color of mycelium to brown to grey black besides
narrowing hyphal width slightly (Table 1). These fungi
also affected the morphology of conidia of V. inaequalis
to a greater extent. T. roseum led to the formation of
single celled and pear shaped conidia measuring
16.52×7.64m instead of 0-1 septate, flame shaped
measuring 22.42×6.65m of V. inaequalis. The other
two fungi viz P. expansum and A. mali caused V.
inaequalis to produce mostly single celled conidia of
obpyriform to clavate shaped measuring 21.92×6.82m
and 22.16×6.74m, respectively. Production of conidia
in dual culture indicated that T. roseum drastically
reduced the conidial production to the extent of 3.5×10 2
spores/ ml compared to 9.2×10 4 spores/ ml in control.
The next best fungus in this regard was P. expansum
(4.8×10 3 spores/ ml) followed by A. mali (7.4×10 3
spores/ ml), respectively. Their effect on delay in
formation of conidia by V. inaequalis indicated T.
roseum did not allow the pathogen to produce conidia
up to 35 days of its growth. However, other two fungi
viz P. expansum, and A. alternata delayed the conidia
production by 21 and 16 days in order (Table 1).
Further, T. roseum inhibited the conidial (harvested
from dual culture) germination by 72.1 per cent
whereas; P. expansum (35.2%), A. mali (24.7%) were
less effective. The morphological characteristics of V.
inaequalis recorded in the present studies are similar to
that observed by Gupta and Lele (1980b).
Effect of test fungi on development of pseudothecia and
ascospores productivity of apple scab pathogen
T. roseum was highly effective in inhibiting both
the formation of pseudothecia on fallen leaves as well
as subsequent ascospores productivity (Table 2). It did
not allow the formation of pseudothecia at its inoculum
density of 2.0×10 4 and 1.5×10 4 cfu/ ml. However, at
lower levels of inoculum density ranging between
0.2×10 4 -1.2×10 4 cfu/ ml; it inhibited development of
pseudothecia between 44.7-94.8 per cent. T. roseum at
three levels of inoculum density (1.2-2.0×10 4 cfu/ ml)
also resulted in complete inhibition of ascospore
productivity of V. inaequalis. Further lowering in the
inoculum density of this test fungus has led to increase
in production of ascospores and it was 30.9 per cent at
the lowest concentration of 0.2×10 4 cfu/ ml. Use of P.
expansum also inhibited the formation of pseudothecia
and subsequent ascospores productivity to some extent
ranging between 18.1-52.2 and 12.6-56.4 per cent,
respectively, whereas A. alternata was least effective
(Table 2). Thakur and Gupta (1991) observed that
amongst 27 fungi isolated from apple phylloplane,
Chaetomium globosum, Athelia sp. and Trichoderma
viride were effective in reducing the development of
pseudothecia production of V. inaequalis on fallen
leaves. The former two fungi have also earlier been
found effective in significantly inhibiting the
development of pseudothecia as well as production of
247

ascospores on fallen leaves by enhancing their
decomposition (Heye and Andrews 1983, Zavara et al.
1994). The present observations indicate that T. roseum
isolated from scab lesions on apple fruit was not only
effective in inhibiting the pseudothecial development
but was also successful in parasitizing the mycelium of
V. inaequalis and appreciably decreasing the production
of ascospores as well as conidia. An inhibition in
development of pseudothecia and subsequent ascospore
I M Sharma
productivity brought by T. roseum may be due to its
capability to parasitize the mycelium of V. inaequalis,
as established in the present studies, as well as its
competition with scab fungus for space on leaf surface
besides enhancing the leaf decomposition. The latter
two facts as above may be true for other two fungi P.
expansum and A. alternata for consequential reduction
in the formation of pseudothecia, and subsequent
production of ascospore of apple scab pathogen.
LITERATURE CITED
Andrews J H, Berbee F M and Nordheim E V. 1983. Microbial antagonism to the perfect stage of apple scab pathogen,
Venturia inaequalis. Phytopathology 73: 228-234
Anonymous. 2003. Package of Practices for Fruit Crops. Published by Dr. Y.S. Parmar University of Horticulture and
Forestry, Nauni, Solan, Himachal Pradesh 186pp
Barnett H L. 1969. Illustrative genera of imperfect fungi. 2 nd Ed., Burgess Publishing Company. 426 S., Sixth Street
Minneapolis IS Minn 225pp
Basu C K C and Puttoo B L. 1984. Fungicide resistant strains of Venturia inaequalis in Kashmir–A prediction. In:
Proceeding British Crop Protection Conference–Pests and Diseases, Brighton (UK) 6A, 12: 509-513
Gomez K A and Gomez A A. 1984. Statistical Procedures for Agricultural Research, II Ed. John Willey and Sons
Publication, New York 680pp
Gupta A K, Sharma I M and Malhotra R. 2000. Cultural variation and carbendazim insensitivity in Venturia inaequalis
isolates from Kullu valley. Journal Hill Research 13: 11-14
Gupta G K. 1978. Present status of apple scab (Venturia inaequalis) in Himachal Pradesh and strategy for its control.
Pesticides 12: 13-14
Gupta G K. 1981. Maturation and discharge of ascospores of Venturia inaequalis. Indian Phytpathology, 34: 502-504.
Gupta G K and Lele V C. 1980a. Role of urea in suppression of ascigerous stage and comparative in vitro efficacy of
fungicides against apple scab. Indian journal Agricultural Sciences 50: 167-173
Gupta G K and Lele V C. 1980b. Morphology, physiology and epidemiology of apple scab fungus, Venturia inaequalis
(Cke.) Wint. in Kashmir Valley. Indian Journal Agricultural Sciences 50: 51-60
Gupta G K and Raj M. 1988. Studies on the factors affecting development of pseudothecia of Venturia inaequalis, the
apple scab pathogen. Plant Disease Research 3: 22-31
Gupta G K and Verma K D. 1986. Studies on keeping quality of apple fruit infected with scab (Venturia inaequalis) in late
summer season. pp307-309. In: Chadha, T.R., bhutani, V. P. and Kaul, J.L. (Eds.). Advances in Research on
Temperate Fruits, Dr. Y.S. Parmar University of Horticulture and Forestry, Solan (HP), India
Gupta G K, Verma K D and Pal J. 1984. Some observations on the prevalence and severity of apple scab in Himachal
Pradesh during the year 1978-1983. Indian Journal of Mycology and Plant Pathology 14: 12-14
Gupta V K and Sharma J N. 1996. Reduced efficacy of carbendazim against Venturia inaequalis causing apple scab. Plant
Disease Research 11: 161-163
Heye C C and Andrews J H. 1983. Antagonism of Athelia bombacina and Chaetomium globosum to the apple scab
pathogen, Venturia inaequalis. Phytopathology 73: 650-654
Huang H and Hoes J A. 1976. Penetration and infection of Sclerotinia sclerotiorum by Ciniothyrium minitans. Canadian
Journal Botany 54: 406-410
Kaul J L. 1985. Storage scab and its management, pp96-98. In: Agarwala, R.K., Kaul, J.L. and Bhardwaj, L.N. (Eds.), Principal
and Concepts of Apple Scab Disease Management. Haryana Agricultural University Press, Hissar, India
Sharma I M. 2004. Integrated management of scab lesions, rots and shriveling of apple fruits during storage. Scientific
Horticulture 9: 79-84
Sharma I M and Bhardwaj S S. 2000. Use of plant extracts and yeast antagonists in the management of storage scab and
rots in apple. Journal Biological Control. 14: 17-23
Sharma I M, Bhardwaj S S and Gupta V K. 1995. Possible factors responsible for the epiphytotic of apple scab in Kullu
valley of Himachal Pradesh during 1995, pp12. In: Proceeding National Symposium on Recent Trends in the
Management of Biotic and Abiotic Stress in Plants organized by Indian Society of Plant Pathologists on Nov. 2-
3, 1995 at CSK HPKV-Palampur (HP), India
Sharma I M and Gupta V K. 1996. Studies on pseudothecial development of apple scab fungus (Venturia inaequalis) in
relation to environmental factors. Plant Disease Research 11: 222
Thakur V S and Gupta V K. 1991. Apple phylloplane microbes as antagonists to Venturia inaequalis. Indian Phytopathology 44: 34
Thakur V S and Sharma R D. 1999. Apple scab and its management, pp54-79. In: L.R. Verma and R.C. Sharma (Eds.).
Diseases of Horticultural Crops-Fruits. Indus Publishing Company, New Delhi, India
Vincent J M. 1947. Distortion of fungal hyphae in the presence of certain inhibitors. Nature 159: 850
Zvara J, Curnova A and Koubova R R. 1994. The effect of antagonistic fungi Athelia bombacina on overwintering stage of
apple scab pathogen Venturia inaequalis. Fytotechnicka-Rada 11: 77-85
248