Romanian Biotechnological Letters
Copyright © 2015 University of Bucharest
Vol. 20, No. 5, 2015
Printed in Romania. All rights reserved
ORIGINAL PAPER
The influence of some abiotic factors on the development
of Alternaria spp. pathogen ("in vitro")
Received for publication, June 10, 2015
Accepted, September 10, 2015
MARDARE E.Ş 1*, CRISTEA S.1, GÂDEA M1, TAMBA-BEREHOIU R.1
1
University of Agronomic Sciences and Veterinary Medicine, 59 Mărăști bd., sect1,
011460, Bucharest,România
*Corresponding author: E-mail:[email protected]
Abstract
Alternaria spp. fungus frequently colonizes sunflower achenes. The aim of the researches was to establish
the biological terms of Alternaria spp pathogen development in vitro conditions. The biological material was
represented by sunflower achenes belonging to Tristan hybrid. It was observed the influence of abiotic factors
(temperature, light and the culture medium) on growth and sporulation of Alternaria spp fungus. The pathogen
was isolated from the sunflower achenes and introduced in the thermostat at temperature of 22°C. The
observations were made at 3, 6 and 9 days after incubation for the experiments regarding the light influence
and the growth substrate and at 3, 6, 9, 12 and 15 days in order to determine the thermal thresholds. The
colony diameter was measured and it was appreciated the presence of fructifications. The optimal thermal
threshold of development for the fungus ranged between 24-26°C, the fungus developing colonies with 70 mm,
with abundant sporulation, after 15 days of observation. In light/dark alternation the fungus Alternaria spp.
had invaded the culture dish and thoroughly exploited.
Keywords: sunflower, achenes, fungus, abiotic factors
1. Introduction
The pathogenAlternaria spp.produces blackish-brown spots on leaves, stems, petioles,
and inflorescence which unite and form large necrotic areas (OLIVEIRA et al [12]; CRISTEA
[5]). Also, the fungus Alternaria spp. is considered an important pathogen of the seed
(UDAYASHANKAR et al [16];CRISTEA(MANOLE) et al [4]; COZEA & CRISTEA [3];
MARDARE et al [11]; PANA et al [13];CRISTEA et al [6]). Primary infections are carried
by through conidia from crop residues that are left on the ground or diseased seeds (RADU et
al [14]). Alternaria spp. micromycete forms characteristic spore features that ensure the
perpetuation and spread of the fungus (GHEOGHIEŞ & CRISTEA [7]). Alternaria
spp.Fungusgrowth and development is determined by temperature, humidity and light
(ALLEN et al [1]). The data from the scientific literature show that on the potato-dextroseagar culture medium, the fungus grows well at 23-25°C (ISLAM&MARIC [9]; ISLAM et al
[10]). Knowing the influence of abiotic factors on the growth and development of plant
pathogens it is important study their biology (CRISTEA S. et al [16], CRISTEA S. et al [17]).
2. Materials and methods
Researches on the influence of abiotic factors (the temperature, light and culture medium)
on the growth and sporulation of Alternariaspp.fungus, were carried out in laboratory
Romanian Biotechnological Letters, Vol. 20, No. 5, 2015
10880
The influence of some abiotic factors on the development
of Alternaria spp. pathogen ("in vitro")
conditions. The biological material was represented by sunflower achenes from Tristan
hybrid. Alternaria spp. Culture was obtained through the isolation and successive subculturing of the fungus on potato-dextrose agar culture medium and incubated at 22°C
(HULEA [8]; RAICU [15]). In order to determine the nutrient substrate which is the most
favorable for the growth and sporulation of Alternaria spp.fungus, were chosen different
nutrient mediums: Potato-dextrose agar, malt agar (medium semi-synthetic), Czapek Dox agar
(synthetic medium), and natural mediums: flour corn-agar, oat flour-agar, rice-extract agar,
wheat-agar, bean-agar (CONSTANTINESCU[2]). The influence of light on the growth and
sporulation of the fungus Alternaria spp. was determined by monitoring the light factor in the
next variations: continue lightening, alternating light/darkness 8h/16h, 12h/12h, 24h/24h and
continuous darkness. The influence temperature on the growth and sporulation of the fungus
was performed on thermostat crops at temperatures between 2 - 400C. Determinations were
performed on the vegetative growth of the fungus by measuring the diameter of the colony
and it was rated the degree of sporulation. The observations were made at 3, 6 and 9 days for
the experiences regarding the influence of culture substrate and lightening and at 3, 6, 9, 12,
15 days the temperature parameter. Data were statistically analyzed.
3. Results and discussion
The temperature has a decisive role in the development of infections and the pathogen's
development. The data in (Table 1) show that the fungus begins to increase at 20C after 9 days
of observation, forming a loose mycelium. The micromycete sporulates weak starting with the
temperature of 80 C. The optimal threshold fot the development of the fungus ranged between
24-260 C, when the fungus formed colonies of 70 mm diameter and abundant sporulation,
after 15 days of observation (Figure.1).At a temperature of 360C growth of the fungus was
slower, and the diameter of the colony reached 32 mm. At a temperature of 400 C the fungus
stopped its evolution. From the statistical point of view is was found that in variants with
temperatures below 24oC and over 32oC was registered a very significant negative reduction
in the colonies' diameter.
Table 1. The temperature's influence of incubation on Alternaria spp. fungus colony size
T0C
/
days
2
4
6
8
10
12
14
16
18
20
22
24
Diameter of colony ( mm)/days
0
0
0
0
0
2
3
3
3
4
4
5
3
-5ooo
-5ooo
-5ooo
-5ooo
-5ooo
-3ooo
-2ooo
-2ooo
-2ooo
-1ooo
-1ooo
0
0
1
1
2
4
12
13
13
14
16
17
18
6
-22ooo
-21ooo
-21ooo
-20ooo
-18ooo
-10ooo
-9ooo
-9ooo
-8ooo
-6ooo
-5ooo
-4ooo
1
6
6
8
10
16
18
18
19
22
25
28
9
-28ooo
-23ooo
-23ooo
-21ooo
-19ooo
-13ooo
-11ooo
-11ooo
-10ooo
-7ooo
-4ooo
-1
3
9
10
11
15
30
25
26
28
29
35
37
12
-35ooo
-29ooo
-28ooo
-27ooo
-23ooo
-8ooo
-13ooo
-12ooo
-10ooo
-9ooo
-3oo
-1
Romanian Biotechnological Letters, Vol. 20, No. 5, 2015
Observations on the
vegetative mass and
sporulation after 15
days
15
5
10
11
16
24
32
32
38
42
48
68
70
-65ooo
-60ooo
-59ooo
-54ooo
-46ooo
-38ooo
-38ooo
-32ooo
-28ooo
-22ooo
-2
0
Vm ±Sp 0
Vm ±Sp 0
Vm ±Sp 0
Vm +Sp±
Vm+Sp +
Vm+Sp+
Vm++Sp+
Vm++Sp+
Vm++Sp++
Vm++Sp++
Vm++Sp++
Vm+++Sp+++
10881
MARDARE E.Ş. CRISTEA S, GÂDEA M.,
TAMBA-BEREHOIU R.
5
26
5
28
5
30
3
32
3
34
2
36
2
38
0
40
0
42
DL 5%
Mt
0
0
-2ooo
-2ooo
-3ooo
-3ooo
-5ooo
-5ooo
0,15
DL 1%
DL 0,1 %
0,26
0,34
22
32
34
28
20
10
6
0
0
Mt
10***
12***
6***
-2ooo
-12ooo
-16ooo
-22ooo
-22ooo
0,85
1,39
1,72
29
40
42
38
25
12
8
0
0
Mt
11***
13***
9***
-4***
-17ooo
-21ooo
-29ooo
-29ooo
1,05
38
50
48
46
30
12
9
0
0
Mt
12***
10***
8***
-8ooo
-26ooo
-29ooo
-38ooo
-38ooo
1,42
1,90
2,46
2,51
3,66
70
68
50
50
32
32
9
0
0
Mt
-2
-20ooo
-20ooo
-38ooo
-38ooo
-61ooo
-70ooo
-70ooo
2,06
Vm+++Sp+++
Vm+++Sp+++
Vm++Sp++
Vm++Sp++
Vm+Sp+
Vm ±Sp ±
Vm ±Sp 0
Without growth
Without growth
3,71
5,09
Legend: mv± = very poor vegetative mass, mv + = poor vegetative mass, mv ++ = good vegetative mass, mv
+++ = very good vegetative mass, Sp+ = poor sporulation, Sp ++ = good sporulation, Sp+++ = abundant
sporulation, Sp ± = very poor sporulation, 0= fungus did not sporulate
Figure 1. The temperature's influence on the growth and sporulation
of Alternaria spp. fungus after 15 days at26°C.
Table 2. The light's influence on the growth and development of Alternaria spp.pathogen
Light/Dark
3
Colony diameter (mm)/days
6
Mt
70
80
Mt
5***
56
-14ooo
80
12***
75
5*
85
75
85
0
5*
9
Observations on the vegetative
mass and sporulation
Vm++ Sp++
Vm++ Sp+++
Vm+++ Sp+++
Vm +++Sp+++
12/12
23
Mt
16/8
28
0
24/24
35
5*
Continuous
23
5*
lightening
Continuous dark
31
8***
45
-25ooo
75
-5o
Vm ++Sp+
DL 5%
1,50
3,86
4,33
DL 1%
2,56
6,34
7,39
DL 0,1 %
3,52
8,42
10,18
Legend: mv++=good vegetative mass,mv +++ = very good vegetative mass, Sp++= good sporulation, Sp+++ =
abundant sporulation, Sp+ = poor sporulation
Figure 2.The influence of light/12/12 on the growth and sporulation of
Alternaria spp fungus after 9 days.
10882
Romanian Biotechnological Letters, Vol. 20, No. 5, 2015
The influence of some abiotic factors on the development
of Alternaria spp. pathogen ("in vitro")
From the analyzed data in (Table 2), Alternaria spp. fungus has developed best under light
and continue alternating light/dark 24/24 conditions, reaching 85 mm diameter colony after 9 days
of observation. In light/dark 12/12, 16/8 alternating, the fungus has developed a diameter of 80
mm after 9 days of observation (Figure 2). Exposed to continuous darkness the fungus has
developed quite well after 3 days of observation, but the growth was slower during the
observations so that after 9 days from the incubation the diameter of the colony was smaller than
the variants that have been exposed to the light. The data were confirmed by statistical analysis.
Table 3.The influence of different culture mediums on Alternaria spp. development
Culture medium
3
Synthetic
mediums
Semisynthetic
mediums
Natural
mediums
DL 5%
DL 1%
DL 0,1 %
Czapek-Dox-agar
26
Potato-dextrose-agar
Malt-agar
28
30
Corn flour agar
Oat flour - agar
Rice-extract agar
Wheat -agar
Beans-agar
10
12
33
37
32
Diameter of the colony (mm)/ days
6
9
39
-2
45
50
Mt
2
ooo
-18
-16ooo
5***
9***
-2
15
25
47
54
48
50
-6
Mt
5
ooo
-30
-20ooo
2
9***
3*
1,46
2,24
2,95
2,43
3,56
4,85
-20
70
68
Mt
25
45
70
70
63
-45ooo
-25ooo
0
0
-2
-7ooo
Observations on
the vegetative
mass and
sporulation
Vm+Sp++
Vm++Sp+++
Vm++Sp++
Vm±Sp ±
Vm++Sp++
Vm+++ Sp+++
Vm+++ Sp+++
Vm+Sp+
3,18
5,12
6,54
Legend: mv ++ = good vegetative mass, mv +++ = very good vegetative mass, mv ± = very poor vegetative
mass, mv + = poor vegetative mass, Sp ++ = good sporulation, Sp+ = poor sporulation, Sp+++ = abundant
sporulation, Sp ± = very poor sporulation
Figure 3. The influence of PDA culture medium on the growth and
sporulation of Alternaria spp after 9 days.
Regarding the influence of the substrate of the culture on the growth of the pathogen, the
data presented in (Table 3) show that the nutritive medium can influence the growth and
sporulation of Alternaria spp. fungus. The medium of culture less favorable to the growth
of Alternaria spp. fungus was the corn flour and agar one, where the diameter of the colony
reached 25 mm after 9 days of observation. The fungus preferred the potato-dextrose agar semisynthetic medium, followed by the natural mediums like rice and agar extract, agar wheat,
developing colonies with a maximum diameter of 70 mm and abundant sporulation (Figure 3).
The observations regarding the culture medium’s influence highlights that the pathogen shows
plasticity on the natural medium of culture. The data obtained are confirmed statistically.
Romanian Biotechnological Letters, Vol. 20, No. 5, 2015
10883
MARDARE E.Ş. CRISTEA S, GÂDEA M.,
TAMBA-BEREHOIU R.
4. Conclusion
Alternaria spp. fungal colonizes often the sunflower achenes presenting a large plasticity
to the analyzed parameters. The fungus has developed optimally at a temperature of 24-280C.
The maximum developmental threshold can be considered 380C and a temperature of
0
40 C was lethal. The light favored the development of Alternaria spp. pathogen.
The potato-dextrose agar semi-synthetic medium was favorable to the growth and
development of the fungus.
Among the natural mediums Alternaria spp. fungus preferred agar rice-extract and wheat
agar substrates.
5. Acknowledgements
This paper was published under the frame of European Social Found, Human Resources
Development Operational Programme 2007-2013, project POSDRU/159/1.5/S/132765
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
10884
ALLEN, S. J., BROWN, J. F., KOCHMAN, J. K. 1983. Effects of temperature dew period and light on
the growth and development of Alternaria helianthi.Phytopathology, 73, 893-896.
CONSTANTINESCU O.1974, Metode şi tehnici în micologie. Ed. Ceres, Bucureşti
COZEA, A.,CRISTEA, S. 2011. Aspects regarding pathogen-enzymatic system interrelation at
Momordica charantia naturalized in Romania. Romanian Biotechnological Letters, 16(5), 6669-6674
CRISTEA (MANOLE), MS, CRISTEA, S.,ZALA, C. 2015. Research on micoflora present in the
caryopses of wheat (Triticum aestivum) in the SE of Romania, in terms of 2014. Romanian
Biotechnological Letters, 20(1), 10183-10189..
CRISTEA S. 2005. Fitopatologie vol. 2, Ed. Cris Book Universal, Bucureşti
CRISTEA STELICA, V. JINGA, C. ZALA, C. POPESCU, S. GLIGA, N. PATRASCU.2009. The
oversee of mycotical rots and chemical influence on their attack on sunflower. Scientific Papers, Series
A, Vol LII: 409-416
GHEORGHIEŞ C., CRISTEA S. 2001. Fitopatologie, vol.1, Ed Ceres, p.135
HULEA ANA. 1969. Ghid pentru laboratoarele de micologie si bacteriologie , Ed. Agrosilvica, Bucuresti
ISLAM, R.,MARIC, A.1978. Alternaria helianthi on sunflower inYugoslavia and somecultural
characteristics of the fungus. Zastita bilja, 29: 145-150
ISLAM, U., MARIC, A., CUK, L., SKORIC, D. 1976. Studies of Alternaria black spot (Alternaria helianthi
Hansf.) of sunflower in Yugoslavia. Proc. 7th Intern. Sunflower Conf., Krasnodar, USSR, Vol. II : 230–237
MARDARE, E. Ş., CRISTEA, S., ZALA, R. C. 2014. Researches On The Micoflora Of Sunflower’s
Achenes For The Hybrids Cultivated In Fetesti Area, Ialomita County. Lucrări Ştiinţifice,USAMV
“Ion Ionescu de la Brad” Iasi Vol 57: 213-216.
OLIVEIRA, M. F. D., NETO, A. T., LEITE, R. M., CASTIGLIONI, V. B., ARIAS, C. A. A. 2004.
Mutation Breeding In Sunflower For Resistance To Alternaria Leaf Spot/ Helia, 27(41), 41-50.
PANĂ, M., CRISTEA, S., CERNAT, S., NEGRILĂ, E. 2014. The Mycoflora Of Barley-The Varieties
Extension Certificated At Ards-Teleorman. Lucrări Ştiinţifice, USAMV “Ion Ionescu de la Brad” Iasi. Vol
57: 217-220
RADU, E., CRISTEA STELICA, C. R. ZALA. 2011.Research on the biological features of Alternaria
brassicae pathogen isolated on rape. Scientific Papers, USAMV Bucharest, Series A 54: 350-355.
RAICU C., BACIU D. 1978. Patologia semintei. Editura Ceres, Bucuresti
STELICA CRISTEA, MARIA OPREA, MIHAELA COSERU.2000.Monilinia laxa ( Aderh) et Ruhl
Honney – Biological parameters of the fungus’s development Lucrari Stiintifice , Seria B, XLIII: 197- 198.
STELICA CRISTEA,MARIA OPREA, MIHAELA COSERU.2000. The influence of certain
biological factors upon the development of the Sclerotinia sclerotiorum fungus ( Lib) de Bary.Lucrari
Stiintifice, Seria B, XLIII: 145-148
UDAYASHANKAR, A. C., NAYAKA, S. C., ARCHANA, B., ANJANA, G., NIRANJANA, S. R.,
MORTENSEN, C. N., PRAKASH, H. S. 2012. Specific PCR-based detection of Alternaria helianthi:
the cause of blight and leaf spot in sunflower. Archives of microbiology, 194(11), 923-932.
Romanian Biotechnological Letters, Vol. 20, No. 5, 2015