TURKISH PHYTOPATHOLOGICAL SOCIETY EDITORIAL BOARD

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TURKISH PHYTOPATHOLOGICAL SOCIETY
EDITORIAL BOARD
President
Vice- President
General Secretary
Treasurer
Editor-in-Chief
Prof. Dr. Mehmet YILDIZ
Dr. Aydan KAYA
Res. Ass. Dr. İsmail Can PAYLAN
Assoc. Prof. Dr. Mustafa GÜMÜŞ
Assoc. Prof. Dr. Pervin KINAY TEKSÜR
SCIENTIFIC REVIEW BOARD
Prof. Dr. Gülay TURHAN
Prof. Dr. Ersin ONOĞUR
Prof. Dr. Nafiz DELEN
Prof. Dr. F.Sara DOLAR
Prof. Dr. Figen YILDIZ
Prof. Dr. Savaş KORKMAZ
Assoc. Prof. Dr. Himmet TEZCAN
Assoc. Prof. Dr. Seral YÜCEL
Assoc. Prof. Dr. Mustafa GÜMÜŞ
Asist. Prof. Dr. Sibel DERVİŞ
Dr. Aydan KAYA
Asist. Prof. Dr. Nazlı Dide Kutluk YILMAZ
Prof. Dr. Zekai KATIRCIOĞLU
Prof. Dr. Berna TUNALI
Assoc. Prof. Dr. Çiğdem ULUBAŞ SERÇE
Prof. Dr. Semih ERKAN
Prof. Dr. Filiz ERTUNÇ
Prof. Dr. Özgür A. KARABULUT
Prof. Dr. Gülsen SERTKAYA
Assoc. Prof. Nuh BOYRAZ
Assoc. Prof. Mine SOYLU
Assoc. Prof. Dr. Semra DEMiR
Assist. Prof. Dr. Ömer ERİNCİK
Asist. Prof. Dr. Nedim ÇETiNKAYA
Prof. Dr. Saadettin BALOĞLU
Assist. Prof. Dr. Arzu COŞKUNTURA
The Journal of Turkish Phytopathology, issued three times a year, is an official
publication of The Turkish Pyhtopathological Society, and publishes original
research papers, reports of new plant diseases and accomplishments.
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ISSN 0378 - 8024
THE JOURNAL OF TURKISH
PHYTOPATHOLOGY
TURKISH PHYTOPATHOLOGICAL SOCIETY
VOL. 38
December
NO. 1-3
CONTENTS
Biological control studies on Convolvulus arvensis L.
with fungal pathogens
B. TUNALI, B. KANSU, D. K. BERNER ...............................................................1
Detection of Seed Borne Mycoflora of Sorghum in Turkey
E. B. TURGAY, F. ÜNAL ......................................................................................9
Activation of systemic disease resistance by acibenzolarS-methyl and a non-pathogen Fusarium oxysporum melonis
(FOM) strain against Fusarium wilt disease in eggplant seedlings
H. H. ALTINOK ...................................................................................................21
The Effect of Charcoal Rot Disease (Macrophomina Phaseolina),
Irrigation and Sowing Date on Oil and Protein Content
of Some Sesame Lines
P. SAĞIR, A. SAĞIR, T. SÖĞÜT .......................................................................33
J. Turk. Phytopath., Vol. 38 No. 1-3, 1-8, 2009
ISSN 0378 - 8024
Biological control studies on Convolvulus arvensis L.
with fungal pathogens
B. TUNALI*
B. KANSU*
D. K. BERNER**
* Ondokuz Mayis University, Agricultural Faculty, Department of Plant Protection, 55139
Atakum, Samsun, TURKEY
** USDA, ARS, FDWSRU,1301 Ditto Avenue, Fort Detrick, MD 21702–5023, USA
ABSTRACT
Field bindweed (Convolvulus arvensis L.) is a perennial, noxious weed in Europe
and in many agricultural areas of the world, including Turkey. Some pathogenic fungi
were identified as potential to control bindweed and some of them could be used as
mycoherbicide components. In the summer of 2008, 2009 and 2010 the diseased
bindweed plants were collected from different sites of Amasya, Ankara, Çorum,
Samsun and Tokat provinces. Pathogenic fungi were isolated from diseased plants and
they were identified based on their morphological characteristics. Bindweed plants were
grown in a climatically controlled room to the 4 to 5-leaf stage; they were inoculated
with an aqueous spore suspension of each fungi at various densities specified. Spores
were sprayed onto bindweed plants with a hand sprayer until runoff. Dates were
recorded for each isolate when disease lesions became visible, and the proportions of
diseased leaves, out of the total number of leaves, on each inoculated plant were
recorded. Stagonospora convolvuli, Colletotrichum linicola and Myrothecium
verrucaria produced the highest level of diseases on the inoculated test plants. Plant
heights of C. arvensis were recorded the shortest following inoculation with C. linicola
and a Phoma sp. These results indicate that, C. linicola seems potentially effective and
field tests alone or in combination with S. convolvuli, should be performed.
Key words: mycoherbicide, field bindweed, biological control, fungi
INTRODUCTION
Field bindweed (Convolvulus arvensis L.) is a perennial, noxious weed in Europe
and in many agricultural areas of the world, including Turkey. It is a serious problem in
1
BIOLOGICAL CONTROL STUDIES ON CONVOLVULUS ARVENSIS L.
WITH FUNGAL PATHOGENS
wheat, maize, vineyards, beans and other vegetables grown in fields. Since this weed is
a deep-rooted perennial, it can survive chemical and mechanical control measures.
Some control can be achieved with herbicides such as 2,4 -D, dicamba, picloram and
imazapyr glyphosate. However, once a bindweed population is established it is very
difficult to control. Repeated applications of herbicide may stop shoot growth and
reduce the amount of root, but even after applications for several years, some roots
grow, from which further shoots can develop (Timmons, 1949). Bindweed produces
numerous seeds, up to 107 per ha, which survive for 20-30 years in the soil (Timmons,
1949). Because of these control difficulties scientists have been moving towards
biological control methods since the 1970s.
Some pathogenic fungi have been identified with potential to control bindweed and
be used as mycoherbicide components. The European COST Action 816 project, a five
year collaboration between scientists from five European countries, made important
contributions to biological control of both field and hedge bindweeds (C. arvensis and
Calystegia sepium). Pfirter et al. (1997) obtained 154 fungal isolates from 28 fungus
genera from bindweeds. Of these, Stagonospora convolvuli, strain LA39, was shown to
have great potential as a bioherbicide for control of field and hedge bindweeds (Défago et
al., 2001). This strain was extensively tested for effectiveness and host specificity in field
trials in different locations and was found to be very effective and environmentally safe.
Phoma exigua from the south of England was found to be sufficiently effective to
be considered as a potential mycoherbicide. In laboratory experiments each strain of P.
exigua was shown to kill seedlings when applied to the three to five leaf stages with 106
conidia/ml. There was no regrowth from the roots (Pfirter, et al., 1997). Phoma proboscis
was also found very effective against bindweeds in the USA (Heiny and Templeton,
1991). One of Colletotrichum linicola isolate (06-21) was found a destructive pathogen on
field bindweeds in a climatic room experiment in Turkey (Tunali et al., 2008). In
controlled environments, Phomopsis convolvulus, a fungus being examined as a
bioherbicide for bindweed, produced 95% reduction in foliage biomass and up to 55%
mortality on bindweed (Morin et al., 1989). This fungus was newly discovered in Canada
(Ormeno-Nunez et al., 1988) and has since been patented as a potential biological control
agent.
The objective of this study was to collect and evaluate fungus pathogens of C.
arvensis from Turkey with the goal of ultimately producing a mycoherbicide for control
of this weed.
MATERIALS AND METHODS
In the summers of 2008, 2009 and 2010 diseased bindweed plants were collected
from different regions of Amasya, Ankara, Çorum, Samsun and Tokat provinces.
Pathogen isolations from diseased plants were performed either by directly transferring
2
B. TUNALI, B. KANSU, D.K. BERNER
surface-disinfested diseased tissue onto moist filter paper or by transferring diseased
tissue onto half strength Potato Dextrose Agar (PDA- Merck) plates. Fungi from these
isolations were identified based on their morphological characteristics. Single spore
isolates of each fungus were prepared in water agar. All fungal cultures were stored in
glass tubes at 4°C in the refrigerator and in 2 ml cryovials in a -85°C deep-freezer. For
inoculum preparation, all fungal isolates were grown on half strength PDA (19 g potato
dextrose and 10 g bacto agar in 1L distilled water). Cultures were incubated at 23±°C in
plastic Petri plates from 5 to 15 days, depending on speed of growth of the fungus
species. These plates were then flooded and repeatedly flushed with 10 ml sterile
distilled water and spores were brushed off and collected. Spore density was determined
with a haemocytometer. In total, 13 isolates used in pathogenicity tests.
C. arvensis seeds were washed under running tap water and seeds were sown in
7 cm diameter plastic pots which were transferred to a controlled environmental room.
Each treatment consisted of 4 replicates, one plant in a single pot. When bindweed
plants were at the 4- to 5-leaf stage, plants were inoculated with an aqueous spore
suspension with 0.1% (v/v) Tween 20 (Sigma) at the various densities specified for each
fungus (Table 1).
Table 1. Fungal isolates and spore concentrations used in pathogenicity tests
Fungus species
Phomopsis convolvuli
Myrothecium verrucaria
Ascochyta sp.
Ascochyta sp.
Ascochyta sp.
Diplodia sp.
Diplodia sp.
Colletotrichum linicola
Bipolaris sp.
Phoma sp.
Stagonospora convolvuli
Gloeosporium sp.
Isolate number
4–9
25–9
26–8
10–14
10–75
28–1
10–08
43–4
9–28
10–20
34–1
9–21
10–39
Spores/ml
1x106
1x107
1x107
1x107
1x107
5x104
1x107
1x107
1x107
3x104
1x107
2x106
1x107
Spores were sprayed onto bindweed plants with a small hand sprayer until
runoff. Distilled water, without spores, was applied to control plants. Pots were then
covered with plastic bags for 48h. Dates were recorded for each isolate when disease
lesions became visible. Pathogens were re-isolated from all diseased plants at the end of
the tests. Other data included the total number of diseased and dead leaves on each
diseased plant and plant height were also collected.
Data were analysed with SAS V7 software and an analysis of covariance model
where fungus species was a classification variable and spore concentration a continuous
covariate. This enabled adjustment of species means for spore concentration.
3
RESULTS AND DISCUSSION
As a result of inoculation of C. arvensis with these and other fungi collected in
this study, significant differences were observed for proportion diseased leaves and
plant heights (Table 2 and 3). The most diseased leaves were produced by inoculation
with Stagonospora convolvuli, Colletotrichum linicola, and Myrothecium verrucaria
(Fig. 2) and (Table 2). Diseases symptoms on C. arvensis caused by some of the fungi
collected in this study are shown in Figure 1.
Figure 1. Symptoms on C. arvensis leaves caused by (A) Ascochyta sp., (B) Diplodia sp., (C) Phoma sp., (D)
M. verrucaria
Figure 2. Symptoms on C. arvensis leaves. (A) Stagonospora convolvuli . (B) P. convolvuli. (C) C. linicola
4
Table 2. Mean proportion diseased and dead leaves on C. arvensis after inoculation with spores of fungus
species. The means are adjusted, through analysis of covariance, for the covariate of spore
concentration, and the probabilities associated with t-tests comparing the means to zero are
indicated (P>|t|).
Proportion diseased leaves
P>|t|1
0.09
NS2
Ascochyta sp.
Diplodia sp.
Bipolaris sp.
Phoma sp.
Gloeosporium sp.
0.33
0.10
0.17
0.38
0.22
0.20
0.56
0.04
0.005
NS
0.031
<0.001
NS
NS
<0.001
NS
-0.18
0.30
0.22
0.39
NS
0.0001
0.03
0.003
Fungus species
Contrasts
C. linicola minus S. convolvuli
C. linicola + S. Convolvuli minus all others
C. linicola minus all others except S. convolvuli
S. convolvuli minus all others except C. linicola
1
Probability of a greater absolute value of t in t-tests comparing the means to zero, i.e., the probability that
the mean or contrast is greater than zero
2
Not significantly different from zero at P<0.05
Although a potentially effective control agent, M. verrucaria produces
macrocyclic trichothecene that are toxins harmful to humans (Millhollon et al., 2003),
this fungus was not evaluated further as a potential mycoherbicide component. Both S.
convolvuli and C. linicola caused significantly more disease symptoms on leaves than
all of the other fungi and there was no significant difference between these two fungi
(Table 2). Surprisingly, because of the putative potential of P. convolvuli (Morin et al.,
1989; Ormeno-Nunez et al., 1988; Kuleci 2009), the isolate of this fungus from Turkey
did not produce significant proportion of diseased leaves nor significant reduction in
plant height (Table 3). Plant heights of C. arvensis were the shortest following
inoculation with C. linicola and a Phoma sp. (Table 3).
However, because of the variability in the non-inoculated water control (Table
3), these plant heights were not significantly different than the control. Inoculation with
S. convolvuli had no effect on reducing plant height. Despite this, S. convolvuli has been
extensively investigated and proven effective as a biological control agent of C.
arvensis (Pfirter, et al., 1997; Défago et al., 2001). However, based on the results from
this study, C. linicola seems at least as potentially effective and field tests with both
fungi, alone and in combination, are planned. The results in Table 2 suggest that the
combination of both fungi might be very effective. Of course, the effectiveness of a
biological control agent can be increased by formulation which should be designed to
5
increase both the efficiency of application and efficacy of the control agent. Different
formulations of both of these fungi remain to be tested as the combination with sublethal dosages of herbicides. Immediate future research on these fungi will be on
conidia maturity, survivability formulation dose response pathogen mixtures and
susceptibility of different ecotypes of C. arvensis.
Table 3. Mean C. arvensis plant heights after inoculation with spores of fungus species. The means are
adjusted, through analysis of covariance, for the covariate of spore concentration.
Fungus species
Plant height (cm)
Standard error
33.5 A*
4.54
26.4 AB
3.68
Ascochyta sp.
28.7 A
2.41
Diplodia sp.
31.5 AC
2.63
21.4 B
2.78
Bipolaris sp.
31.4 AB
5.94
Phoma sp.
21.6 BC
3.68
33.8 A
4.24
Gloeosporium sp.
26.4 AB
3.68
Non-inoculated control (water)
31.7 AB
5.95
*Means followed by the same letter are not significantly (P<0.05) different
ÖZET
FUNGAL PATOJENLER ILE CONVOLVULUS ARVENSIS L.’ İN
BİYOLOJİK MÜCADELESİ ÇALIŞMALARI
Tarla sarmaşığı (Convolvulus arvensis) çok yıllık zararlı bir yabancı ot olup
gerek Avrupa’da gerekse Türkiye’de pek çok tarım alanında bulunmaktadır. Tarla
sarmaşığıyla mücadelede potansiyel bir mikoherbisit olarak bazı patojen fungus türleri
saptanmıştır. Hastalıklı tarla sarmaşığı bitkileri 2008, 2009 ve 2010 tarihlerinde, Amasya,
Ankara, Çorum, Samsun ve Tokat yörelerinden toplanmıştır. Hastalıklı bitkilerden
patojenlerin izolasyonları yapılmış ve morfolojik karakterlerine dayanarak fungusların
teşhisleri de yapılmıştır. Tarla sarmaşığı bitkileri iklim odasında 4–5 yapraklı devreye
gelene kadar yetiştirilmiş her bir fungus için farklı yoğunluklarda spor süspansiyonları
hazırlanmıştır. Küçük spreyler kullanılarak sporlar tarla sarmaşığı bitkilerine, bitkileri
tamamen ıslatmak suretiyle püskürtülmüştür. Her bir izolatta hastalık belirtisi oluşma
tarihleri kaydedilmiştir. Veriler SAS istatistik programı, kovarians modeli ile analiz
edilmiştir. Patojenisite testi sonucunda en fazla hastalık Stagonospora convolvuli,
Colletotrichum linicola ve Myrothecium verrucaria ile inokule edilen bitkilerin
yapraklarında saptanmıştır. Bitki boylarında en fazla kısalma C. linicola ve bir Phoma
sp. izolatı ile bulaştırılan bitkilerde olmuştur. Bu sonuçlara göre, C. linicola’ nın etki
6
potansiyeli olduğu görülmektedir. Bu fungusla ilgili tarla denemeleri tek olarak veya S.
convolvuli ile birlikte en kısa zamanda yapılmaya başlanmalıdır.
Anahtar kelimeler: Tarla sarmaşığı, Biyoherbisit, Funguslar, Biyolojik
mücadele
LITERATURE CITED
Défago G., Ammon Hu., Cagan L., Draeger B., Greaves Mp., Guntlı D., Hoeke D.,
Klımes L., Lawrıe J., Moënne-Loccoz Y., Nıcolet B., Pfırter Ha., Tabacchı R.
and Toth P, 2001. Towards the biocontrol of bindweeds with a mycoherbicide.
Biocontrol 46 (2): 157–173.
Heiny, D. K. and Templeton, G. E., 1991. Effects of Spore Concentration, Temperature
and Dew Period on Disease of Field Bindweed Caused by Phoma proboscis.
Phytopathology 81: 905–909.
Kuleci, E., B. Tunali, D. K. Berner, C. A. Cavin, And L. A. Castlebury. 2009. First
report of leaf anthracnose caused by Phomopsis convolvuli on field bindweed in
Turkey. Plant Disease 93: 847.
Millhollon, R. W., Berner, D. K., Paxson, L. K., Jarvis, B. B. and Bean, G. W., 2003.
Myrothecium verrucaria for Control of Annual Morningglories in Sugarcane.
Weed Technology. Volume 17, Issue 2. pp. 276–283.
Morin, L., A. K. Watson and R. D. Reeleder, 1989. Efficacy of Phomopsis convolvulus
for control of field bindweed (Convolvulus arvensis). Weed Science 37: 830–
835.
Ormeno-Nunez, J., R. D. Reeleder and A. K. Watson, 1988. A new species of
Phomopsis recovered from field bindweed (Convolvulus arvensis). Canadian
Journal of Botany 66: 2278-2233.
Pfirter, H. A., Ammon, H. U.,Guntli, D., Greaves, M and P., Défago G., 1997. Towards
the management of field bindweed Convolvulus arvensis) and hedge bindweed
Calystegia sepium) with fungal pathogens and cover crops. Integrated Pest
Management Reviews 2: 61–69.
Timmons, F.L, 1949. Duration of viability of bindweed seed under field conditions and
experimental results in the control of bindweed seedlings. Agronomy Journal 41:
130–133.
Tunali, B., D. K. Berner and H. J. Dubin. 2008. First report of leaf spot caused by
Colletotrichum cf. linicola on field bindweed in Turkey. Plant Disease 92: 316.
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ISSN 0378 - 8024
Detection of Seed Borne Mycoflora of Sorghum in Turkey
Emine Burcu TURGAY
Filiz ÜNAL
Plant Protection Central Research Institute, 06172, Yenimahalle, Ankara, Turkey
E-mail: [email protected]
ABSTRACT
Seed borne mycoflora of 23 sorghum seed samples collected from different
localities of Turkey was investigated using blotter, agar plate and deep freezing methods
as recommended by ISTA. 19 species (Absidia sp, Acremoniella sp., Alternaria
alternata, Aspergillus flavus, Aspergillus niger, Aspergillus sp., Cladosporium sp.,
Curvularia lunata, Drechslera tetramera, Epicoccum sp., Fusarium avenaceum,
Fusarium nygamai, Fusarium proliferatum, Fusarium semitectum, Fusarium
subglutinans, Fusarium verticillioides, Penicillium spp., Rhizopus sp.) of 23 sorghum
seed samples were determined to be new records for Turkey which belong to 11
genera. Our results showed that Alternaria alternata was the predominant species
among these areas. Higher number of fungi was isolated by using deep-freezing method
as compared to agar and blotter methods.
Key words: Sorghum, seed pathology, mycoflora
INTRODUCTION
Sorghum (Sorghum bicolor L. Moench) is the world’s fifth major cereal crop
after wheat, rice, maize and barley (Fageria et al., 1997; Ayana and Bekele, 2000;
Mullet et al., 2002; Mariscal-Landina et al. 2004). Sorghum, Sudangrass (Sorghum
sudanense (Piper.) Stapf) and Sorghum x sudangrass hybrid cultivars are important
fodder plants grown as the second crop for pasture, silage, green chop and hay in
Turkey. Grain sorghum is commonly consumed as foodstuff in developing or less
developed countries (Fageria et al., 1997; Kenga et al. 2004) and it is also used as
forage and raw material in the industries of developed countries (Delciotti et al. 1998;
Kenga et al., 2004). Since sorghum is more tolerant to extreme hot conditions, it has
been replaced with corn most regions in the world (Güler et al., 2008).
9
DETECTION OF SEED BORNE MYCOFLORA OF SORGHUM IN TURKEY
Sorghum is known to suffer from more than 30 fungal diseases (USDA, 1960).
Important seed borne fungal diseases recorded on sorghum are stalk rot (Aspergillus
niger), target spot (Bipolaris sorghicola), stalk rot/anthracnose/red leaf (Colletotrichum
graminicola), seed rot /stalk rot (Fusarium verticillioides), seedling blight/charcoal rot
(Macrophomina phaseolina) and covered smut/grain smut (Sphacelotheca sorghi)
(Richardson, 1990). Seed is the most important input for crop production. Pathogen free
healthy seed is urgently needed for desired plant populations and good harvest. Many
plant pathogens are seed borne and can cause enormous crop losses. Besides, the mold
fungi growing on the seed substratum produce mycotoxins which are hazardous to man
and animals (Halt, 1994).
In Turkey, it became necessary to study mycoflora of the sorghum by the fact
that seed borne Bipolaris spicifera (Syn: Drechslera tetramera) was found to cause a
disease observed in sorghum growing areas in Sakarya Region (Ünal et al. 2010).
Therefore the aim of present study was to reveal seed mycoflora of the sorghum grown
in Turkey.
Totally 23 Sorghum bicolor (L.) Moench, Sorghum sudanense (Piper) Stapf and
Sorghum bicolor x Sorghum sudanense hybrid samples including 10 varieties (Akdarı,
Ogretmenoglu 77, Beydarı, Early sumac, Rox, Leoti, Sugar Graze, Greengo, Gozde 80,
Jumbo) were obtained from four sorghum growing areas in Antalya, Adana, Sakarya,
and İzmir provinces in Turkey. Out of 23 seed samples, 7 were collected from Antalya,
6 from Sakarya, 6 from Adana and 4 from İzmir. Three different methods recommended
by International Seed Testing Association (ISTA) (Anonymous, 1993); Blotter, Deep
Freezing and Agar Plate methods were used on each sample. As a pre-treatment, 10g of
seed sub-samples were surface sterilized for 1 min with 1% sodium hypochlorite
solution (NaOCl).
Blotter Method
Surface sterilised twenty five seeds were placed on three layers of moistened
blotters in each Petri dish 10 cm in diameter. The dishes were then incubated at a
constant photoperiod (12 h day and 12 h night) for 7 days at 20o C and examined under
a stereomicroscope for seed borne mycoflora. Two hundred seeds per sample were
tested for blotter method.
Deep Freezing Method
Twenty five seeds per plate were placed on three layers of moistened blotters.
Seeds were incubated at a constant photoperiod (12 h day and 12 h night) at 20o C for a
day and frozen at -20 oC for 24h. The plates were then kept at 22 ± 1 o C for 5 days.
Two hundred seeds per each sample were tested for deep freezing method.
10
E.B. TURGAY, F. ÜNAL
Agar Plate Method
The seeds were plated on potato dextrose agar (PDA), 10 seeds per Petri dish and
dishes were incubated at 24 oC for 7 days. One hundred seeds from each sample were
tested for agar plate method.
Identification of fungal species
Fungi grown on seeds were identified by using morphological criteria of Ellis (1971),
Hanlin (1990) and Burgess et al. (1994). In order to identify Fusarium spp. subcultures were
made on Carnation Leaf Agar (CLA) and Potato Dextrose Agar (PDA) and incubated at
25oC for 5–7 days. Final identification was made following Leslie and Summerell (2006).
RESULTS
The results of mycological tests indicated that a total number of 11 genera and 19
species of fungi; Absidia sp, Acremoniella sp., Alternaria alternata, Aspergillus flavus,
Aspergillus niger, Aspergillus sp., Cladosporium sp., Curvularia lunata, Drechslera
tetramera (Syn; Bipolaris spicifera), Epicoccum sp., Fusarium avenaceum, Fusarium
nygamai, Fusarium proliferatum, Fusarium semitectum, Fusarium subglutinans,
Fusarium verticillioides, Penicillium spp., Rhizopus sp. were isolated from sorghum
seeds. All of the 19 species isolated from sorghum seeds are new records for Turkey.
The results of the seed tests indicated that 17 fungal species belonging to 9
genera (Alternaria alternata, Aspergillus flavus, Aspergillus niger, Aspergillus sp.,
Cladosporium sp., Curvularia lunata, Drechslera tetramera (Syn; Bipolaris spicifera),
Epicoccum sp., Fusarium avenaceum, F. nygamai, F. proliferatum, F. semitectum,
F. subglutinans, F. verticillioides, Penicillium spp., Rhizopus sp.), and 16 fungal species
in 10 genera (Absidia sp, Alternaria alternata, Aspergillus flavus, Aspergillus niger,
Aspergillus sp., Cladosporium sp., Curvularia lunata, Drechslera tetramera (Syn;
Bipolaris spicifera), Epicoccum sp., Fusarium semitectum, F. subglutinans,
F. verticillioides, Penicillium spp., Rhizopus sp.) were identified by blotter and agar
plate methods respectively. On the other hand deep freezing method allowed us to
identify all 19 fungal species 11 genera clearly.
The seed test performed on total 23 seed samples which were provided from four
different regions where grain sorghum is intensively cultivated, showed that the highest
number of fungi was obtained from Antalya with 17 fungal species (Table 1) followed
by Adana and Sakarya Regions with 11 fungal species (Table 2 and 3) and finally Izmir
Region with 12 fungal species (Table 4). The common fungal species, which were
identified by three seed tests performed on 7 sorghum seeds of Antalya Region
Alternaria alternata, Rhizopus sp., Fusarium semitectum, F. subglutinans and
F. verticillioides (Table 1). The common species identified from the seed samples of
Sakarya were Alternaria alternata ve Drechslera tetramera (Table 3). Alternaria
alternata was the most common fungal species identified from the seeds provided from
all regions (Table 2 and 4).
11
12
NIF = Numbers of infected grains with fungi in seven varieties, % percentage =%P
NIS= Numbers of infected samples out of seven tested.
I% = Percentange of infected seed, ± SD = Standard deviation
Table 1. Numbers and percentages of fungi in infected seeds of sorghum in Antalya region studied by three different methods.
Table 2. Numbers and percentages of fungi in infected seeds of sorghum in Adana region studied by three different methods.
13
14
Table 3. Numbers and percentages of fungi in infected seeds of sorghum in Sakarya region studied by three different methods.
Table 4. Numbers and percentages of fungi in infected seeds of sorghum in İzmir region studied by three different methods.
15
Overall results indicated that Fusarium was dominant species in Antalya while
Drechslera tetramera was commonly identified from Sakarya region. Aspergillus niger
and Fusarium verticillioides which are seed-born and cause stalk rot and seed rot
diseases were found to be common on the seeds collected from Antalya Region.
Important sorghum seed diseases, target spot (Bipolaris sorghicola), stalk
rot/anthracnose/red leaf (Colletotrichum graminicola), seedling blight/charcoal rot
(Macrophomina phaseolina) and covered smut/grain smut (Sphacelotheca sorghi),
(Richardson, 1990) were not detected in the present study. Among 10 different sorghum
seed species, the highest number of different fungal species was identified from Early
sumac providing 11 fungal species. However the least fungal infection was observed in
Greengo (only 3 species) provided from Sakarya.
DISCUSSION
Of the three methods compared in the present study, the deep freezing method
yielded the highest number of fungi (11 genera and 19 species; Absidia sp,
Acremoniella sp., Alternaria alternata, Aspergillus flavus, Aspergillus niger,
Aspergillus sp., Cladosporium sp., Curvularia lunata, Drechslera tetramera,
Epicoccum sp., Fusarium avenaceum, Fusarium nygamai, Fusarium proliferatum,
Fusarium semitectum, Fusarium subglutinans, Fusarium verticillioides, Penicillium
spp., Rhizopus sp.). The deep freeze method seemed to be more effective for the
detection of deep seated as well as slow growing seed borne fungi like Drechslera spp.,
Fusarium spp., Penicillium spp., Alternaria alternata (Niaz and Dawar 2009). Previous
studies on sorghum seeds also revealed that deep freezing method was most suitable for
detection of Fusarium species (Mathur et al. 1975; Nahar et al. 2005). In the present
study, Fusarium species and Drechslera tetramera were isolated in higher percentages
both by blotter and deep freezing method. On the other hand, in this study, saprobic
fungi; Aspergillus flavus, Aspergillus niger, Aspergillus sp. and Curvularia lunata were
isolated in higher percentages by agar plate method. The agar plate method was found
to be the most suitable method for the isolation of saprobic fungi. Mathur and
Neergaard, (1970) and Khan et al. (1988) preferred agar plate method rather than blotter
method for the isolation of Curvularia spp. and Drechslera spp., from seeds of rice.
However, in the present study, Drechslera tetramera and Fusarium species were
isolated in higher percentage by deep freezing method. Alternaria alternata was
identified in highest rates with all methods used in the study.
A lot of fungi isolated in the present study are known to produce mycotoxins.
Mycotoxins have been implicated as having toxic effects on animals and human being
and they can cause severe damage to liver, kidney and nervous system even in low
dosages (Rodricks 1976).
16
Fusarium species (F. avenaceum, F. nygamai, F. proliferatum, F. semitectum,
F. subglutinans, F. verticillioides) obtained from sorghum seeds in this study are known
to produce mycotoxins deoxyninalenol (DON) zearalenone, fusaric acid and
trichothecene. (ApSimon et al., 1990; Miller, 1995; Sweeney and Dobson, 1998; Abbas
et al., 1999; Benneth and Klich, 2003; Desjardins, 2006), Aspergillus flavus produces
aflotoxin, B1, B2, G1 and G2. Alternaria alternata produces alternariols. These toxins
are very toxic and carcinogenic. They may cause liver cancer in human and livestock
animals and especially loss of weight in cattle, pigs and poultry resulting in economic
losses for the farmers (Diener and Davis, 1969; Purchase, 1974; Pesta and Bonday,
1990).
To the best of our knowledge, this is the first report on the mycoflora of
Sorghum seed grown in Turkey. Pathogenic fungal species (A. alternaria, Drechslera
tetramera, Fusarium spp.) were recovered in significant rates. Various fungus species
which were commonly determined in this study, such as Alternaria alternata,
Aspergillus spp. and Fusarium spp. are also known as mycotoxin producers and
dangerous for human and animal health.
ACKNOWLEDGEMENT
We thank Prof. Dr. Salih MADEN for his valuable support on the identification
of fungal species. We would also like to thank Prof. Dr. F. Sara Dolar for her helpful
comments on the original manuscript.
ÖZET
TÜRKİYE’DE SORGUMDA TOHUM KAYNAKLI
MİKOFLORANIN BELİRLENMESİ
Türkiyenin farklı bölgelerinden toplanan 23 sorgum tohum örneğine ait mikoflora
ISTA tarafından önerilen agar, deep-freezing ve blotter yöntemleri kullanılarak araştırılmıştır. 23 sorgum tohum örneğinin 11 genusa ait 19 türü (Absidia sp, Acremoniella sp.,
Alternaria alternata, Aspergillus flavus, Aspergillus niger, Aspergillus sp., Cladosporium
sp., Curvularia lunata, Drechslera tetramera, Epicoccum sp., Fusarium avenaceum,
Fusarium nygamai, Fusarium proliferatum, Fusarium semitectum, Fusarium
subglutinans, Fusarium verticillioides, Penicillium spp., Rhizopus sp.) Türkiye için yeni
kayıt olarak belirlenmiştir. Elde edilen sonuçlar Alternaria alternata’nın en yaygın tür
olduğunu göstermiştir. Deep-freezing yöntemi ile izole edilen fungus sayısının diğer
yöntemler kullanılarak elde edilen fungus sayısından daha yüksek olduğu belirlenmiştir.
Anahtar kelimeler: Sorgum, tohum patolojisi, mikoflora
17
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ISSN 0378 - 8024
Activation of systemic disease resistance by acibenzolar-S-methyl and a
non-pathogen Fusarium oxysporum melonis (FOM) strain against
Fusarium wilt disease in eggplant seedlings*
H. Handan ALTINOK**
* This study is a part of PhD thesis, accepted by Science Institute of Çukurova University in 2006
** Department of Plant Protection, Faculty of Agriculture, Erciyes University, 38039-Kayseri, Turkey
ABSTRACT
The plant defence activator acibenzolar-S-methyl (ASM; Actigard 50 WG) and a
non pathogenic Fusarium oxysporum strain (FOM; Fusarium oxysporum f. sp. melonis)
were assayed on eggplant seedlings for its ability to induce resistance against Fusarium
oxysporum f. sp. melongenae. Pre-treatment of eggplants with ASM and FOM
significantly reduced the severity of the disease. The lowest disease ratings were
detected at a time interval of 72 h between treatment and pathogen inoculation. With
this interval, disease severity in ASM and FOM-treated plants was reduced to 30.1%
and 20.1%, respectively, while positive control was 91.0%, at 21th day after inoculation.
Microscopical studies showed a strong correlation between the interval of inducerpathogen inoculation and lignin accumulation in xylem cells undergoing hypersensitive
reactions, whereas no staining were observed in negative control plants. Intense lignin
accumulation in xylem vessels indicate both treatments are able induce resistance in
eggplant against this disease.
Key Words: Fusarium Wilt, Induced Resistance, Non-pathogen Fusarium
oxysporum, ASM, Lignin, Eggplant
INTRODUCTION
Fusarium wilt, caused by Fusarium oxysporum Schlecht. f. sp. melongenae, is
one of the most important disease of eggplants in especially Mediterranean Region of
Turkey which lead to serious yield losses. Under optimal infection conditions, this
soilborne pathogen can create necrotic areas by colonizing xylem tissues of susceptible
varieties which blocks water and nutrient transfers, resulting death of the plant (Altınok,
2005). Due to the high inoculum density, control of soil-borne plant pathogens are
21
ACTIVATION OF SYSTEMIC DISEASE RESISTANCE BY ACIBENZOLAR-S-METHYL
AND A NON-PATHOGEN FUSARIUM OXYSPORUM MELONIS (FOM) STRAIN AGAINST
FUSARIUM WILT DISEASE IN EGGPLANT SEEDLINGS
extremely difficult. Soil disinfestation and fungicide application have been commonly
used for controlling these pathogens. There are several studies worldwide on
identification of sources of resistance against Fusarium wilt disease on eggplant but
currently there are no resistant varieties reported. Pathogen’s physiological races are yet
to be defined and there is only one record of vegetative compatibility group (VCG) on
literature (Katan, 1999). It was reported that vegetative compatibility group (VCG0320) of Turkey isolates, and were found compatible with European VCGs (Altınok and
Can, 2010).
In recent years, many methods have been developed for biocontrol of soil borne
plant pathogens by the researchers. Studies on biological control of F. oxysporum f. sp.
melongenae (Fomg) in eggplant are very limited. In plant protection, antibiosis,
competition and hyperparasitism are primary antagonistic mechanisms on controlling
the diseases with biotic factors. Biological control including beneficial microorganisms
is an alternative method for protection of Fusarium wilt diseases (Alabouvette and
Couteaudier 1992). Certain microorganisms can be also protected plants by inducing
plant defense mechanisms, along with their antagonistic efficiency, for supression of
fungal diseases (Kuć, 1982; Matta, 1989). Today, several non-pathogenic bacteria and
fungi biopreparations are in use as successful antagonistic agents against many plant
diseases. Induced resistance as a natural defense mechanism of plants is one of the
ecologically-friendly approaches for plant protection. This inducible defence
mechanism as named systemic acquired resistance (SAR) is effective against many
virulent plant pathogens including, bacteria, fungi and viruses (Ryals et al., 1994). Some
chemicals such as salicylic acid (SA), β-amino butyric acid (BABA), 2,6dichloroisonicotinic acid (INA) with no direct antimicrobial activity can also induce
SAR as well as biotic inducers in plants (Hammerschmidt and Kuć, 1995; Lawton et al.,
1996; Oostendorp et al., 2001). SAR is associated with its several cellular defence
responses including pathogenesis-related (PR) proteins, synthesis of different defencerelated enzymes, rapid and transient production of active oxygen species (AOS),
phytoalexins (Benhamou and Belanger, 1998). The plant defence activator acibenzolarS-methyl (benzo [1,2,3] thiadiazole-7-carbothioic acid-S-methyl ester, ASM) is a
systemic compound used for the control of many fungal and bacterial diseases in
vegetables (Cole, 1999; Elmer, 2006). The compound has minimum antifungal and
bacterial activity, but induces host plant resistance by triggering a natural systemic
activated resistance (SAR) response found in most plant species. This chemical inducer
may be phytotoxic when applications made in the early stage of plants (Conrath et al.,
2001).
Pre-treatment of susceptible plants with avirulent pathogens may induce
resistance to pathogen attack (Kuć, 1982). Several reports have documented the
inducible mechanism to Fusarium wilt by using nonpathogenic strains of F. oxysporum
(Fuchs et al., 1997; Bora and Özaktan, 1998) or formae speciales of F. oxysporum such
22
H. ALTINOK
as f. sp. melonis in cucumber (Gessler and Kuć, 1982) and f. sp. dianthi in tomato
(Kroon et al., 1991). Induced resistance requires that the plant be exposed to the
inducing agent prior to the attack by the pathogen. The time needed for development of
induced resistance is in the range of one to a few days in the case of Fusarium wilt
diseases (Matta, 1989). The role of pathogen-induced lignins and related polymers has
been closely correlated with the defence responses of several plants. Some of the most
extensive work has dealt with lignification in graminaceous plants including reed canary
grass and wheat (Vance et al., 1976). Lignin or the lignification process may role in
plant defence against infection by mechanical barriers to pathogen attack, increasing the
resistance of plant cell walls to the diffusion of toxins from the pathogen and plant cell
wall to be more resistant pathogen cell-wall degrading enzymes (Ride, 1978).
The objective of this work was to test the plant defence activator Acibenzolar-Smethyl (benzo [1,2,3] thiadiazole-7-carbothioic acid- S-methyl ester, ASM; Actigard 50
WG, Syngenta Crop Protection, Inc., Basel Switzerland) and Fusarium oxysporum
formae speciales nonpathogenic on eggplant (FOM; Fusarium oxysporum f. sp.
melonis) and for its ability to induce resistance in eggplants against Fusarium wilt in
climatize conditions.
Plant material
Eggplant seedlings (Solanum melongena L. cv. “Pala”) with four fully expanded
leaves were used for pot experiment. Plants were grown in pots (8.5 cm diam) in a soil
mix containing sand, perlite, and peat compost in the greenhouse and kept in growth
chambers (25°C, 60-70% RH, 12-h photoperiod, 50 to 60 Klux m-2). Seedlings were
watered daily and fertilized with NPK (15:15:15).
Application of ASM
ASM was dissolved in distilled water to obtain a concentration of 0.2 mg ml-1
and then sprayed twice to eggplant seedlings. Fomg10 (Fusarium oxysporum f. sp.
melongenae) as the most virulent isolate by means of DS in a former study were
selected to pathogen inoculation (Altınok and Can, 2010). In order to determine the
optimum time interval for SAR induction, seedlings were first treated with ASM and
then inoculated with the pathogen (Fomg10) suspension 24 h, 48 h, 72 h or 96 h after
treatment using the root-dip assay modified from that of Biles and Martyn (1989).
Pathogen inoculum consisted of spore suspension obtained from one-week-old culture
on Potato Dextrose Agar (PDA; Merck, Germany) and each seedling with wounded
roots was submerged for 10 min, with 100 ml of the conidial suspension (1×106 conidia
ml-1 in sterile H2O), while control plants were sprayed with sterile distilled water instead
of ASM.
23
Inoculation of non-pathogen Fusarium oxysporum
A Fusarium oxysporum formae speciales nonpathogenic on eggplant (FOM;
Fusarium oxysporum f. sp. melonis) was used as a biotic inducer in pot experiments.
Pathogen and inducer fungus were cultured on PDA and Fusarium minimal medium
(FMM); for 7 days in the dark at 25 °C (Nelson et al., 1983). Eggplants were inoculated
with the pathogen (Fomg10) 24 h, 48 h, 72 h or 96 h after FOM (106 spore ml-1)
treatment. Control plants were dipped with sterile distilled water instead of FOM
suspension. After pathogen inoculation the seedlings were transplanted into plastic pots
and kept in a growth chambers as described above.
Disease Assessment
Disease symptoms development was assessed at 7th, 11th, 14th, 17th and 21th day
after inoculation (DAI) with a Fusarium yellow rating of 0 to 4, in which 0 = no lesions,
1 = slight leaf chlorosis and necrosis, 2 = vein clearing on outer leaflets, 3 = yellowing
and dropping of leaves, 4 = dead plant. Plants were evaluated individually and a mean
percent disease severity index (DSI%) was calculated for each assessment day based on
the scale values, according to Townsend-Heuberger formula below (Townsend and
Heuberger, 1943).
In the formula, P; Percentage of disease severity, n; Number of plants in the
disease scale, v; Numerical value of disease score, Z; Highest score number, N; Total
number of plants. The data were subjected to an analysis with Levene’s homogenity of
variance test then grouped by Duncan’s multiple range test (P ≥ 0.05) contained in the
SPSS software (SPSS Inc., Chicago, IL, USA). Both ASM and FOM experiments were
conducted with three replicates of 10 seedlings and repeated twice, representative result
of one experiment for each is presented.
Staining for lignin
Microscopic examination of infected vascular bundles with pathogen was carried
out on a microscope (Nikon Optiphot), equipped with differential interference contrast
(DIC). Photomicrographs were taken using Kodak Gold 200 ISO print film and
developed commercially.
General tissue clearing
Infected vascular bundles were detached from plant then transferred to 100%
methanol, and incubated overnight at room temperature, to remove chlorophyll,
followed by soaking in a aqueous solution saturated with chloral hydrate (2.5 g ml-1) for
24
H. ALTINOK
12-24 h, to soften and clear the tissue (Soylu, 2006). Finally the vascular bundles were
mounted in 50% glycerol and a cover-slip placed over the samples to produce semipermanent preparations.
Lignified structures were visualized using the phloroglucinol/HCl test. Infected
vascular bundles were incubated in a solution of 1% phloroglucinol in 100% methanol
overnight. Following further incubation of cleared tissues in chloral hydrate, they were
subsequently mounted on slides, a few drops of concentrated hydrochloric acid were
added and finally the tissues were covered with a cover slip. After ~10 min, lignified
xylem appeared reddish-orange, but colour faded within ~2-4 h, therefore, preparations
were examined immediately (Gahan, 1984; Vallet et al.,1996; Soylu, 2006).
The effects of the ASM and FOM treatments to Fusarium wilt disease
The time between initial treatment with ASM and FOM significantly protected
the efficacy of induced resistance against damage from subsequent inoculation with
Fomg10. All interval times drastically reduced the DS, but the greatest efficiency by
means of disease suppression was achieved with ASM (Figure 1) and FOM treatment
(Figure 2) 72 h before inoculation. In induced resistance experiment to both inducers
and control plants, the initial symptoms appeared one week after inoculation as
yellowing of the older leaves. The mean DS in control plants was 11.2% (ASM) and
12.3% (FOM) at 7 DAI. The systemic progress of the disease in control plants increased
with time and by 21 DAI, browning areas were observed in the xylem of infected stems.
Eventually, most of the plants collapsed and died. In 72 h inoculations, the mean DS in
control plants reached 91% whereas ASM and FOM-treated seedlings were only 30.1%
and 20.1%, respectively. Resistance induced in eggplant seedlings by ASM and FOM
can be distinguished in Figure 1 and Figure 2. Since the lowest disease ratings were
detected at a time interval of 72 h between treatment and pathogen inoculation, this
interval was taken into consideration in order to detect lignin deposition in both of the
pot experiments.
The determination of phenolics and lignin compounds that accumulate during
infection has been based mainly on microscopy and histochemistry as described by
Nicholson and Hammerschmidt (1992), lignin deposition in infected vascular bundles was
demonstrated histochemically in this study as well. The stain was used here
(phloroglucinol) in the presence of HCl, reacts with aromatic aldehydes, such as
cinnamaldehyde, present in lignins and exhibits a bright reddish-pink colour in infected
plant tissue. Results indicate a strong correlation between the timing and extent of cell
death and high levels of lignin accumulation in cell-walls and cytoplasm of cells
undergoing HR, whereas no staining of lignin-like material was observed during the
25
compatible interaction. The results showed that a significant proportion observed in
eggplant vascular bundles undergoing HR is associated with rapid accumulation of ligninlike compounds indicated with phloroglucinol-HCl staining at sites by 11 DAI (Figure 3).
Figure 1. Effect of acibenzolar-S-methyl (ASM) treatment on the severity of Fusarium wilt disease caused by
Fusarium oxysporum f. sp. melongenae (Fomg). After treatment with ASM or water (control),
eggplant seedlings were inoculated 24, 48, 72, 96 h later with the Fomg10 isolate. Inoculated
seedlings were scored at 7, 11, 14, 17 and 21th day after inoculation using 0-4 scale. A mean disease
severity was calculated from each treatment. Error bars indicate ±1 standard error of the mean.
Figure 2. Effect of non-pathogen Fusarium oxysporum (FOM) treatment on the severity of Fusarium wilt
disease caused by Fusarium oxysporum f. sp. melongenae (Fomg). After treatment with FOM or
water (control), eggplant seedlings were inoculated 24, 48, 72, 96 h later with the Fomg10 isolate.
Inoculated seedlings were scored at 7th, 11th, 14th, 17th and 21th DAI using 0-4 scale. A mean disease
severity was calculated from each treatment. Error bars indicate ±1 standard error of the mean.
26
H. ALTINOK
a
b
d
e
c
f
th
Figure 3. Localization of lignin-like compounds in the xylem of inoculated plants at 11 DAI. The eggplant
seedlings inoculated with the Fomg10 isolate 72 h after ASM and FOM treatments. In (a) and (b),
no staining in vascular bundles (control plant). The site of lignification seen as reddish-orange
coloration, is localized only in xylem vessels (arrow) of ASM-treated plants (c and d). Similarly,
accumulation of lignin-like compounds in xylem of FOM-treated plants (e and f).
The results show the inhibitory effects of the plant activator ASM and nonpathogenic Fusarium oxysporum strain on the disease development by Fomg. Both
inducers induced important levels of disease resistance in eggplant seedling. Similarly, a
27
nonpathogenic strain of F. oxysporum protected cucumber against Fusarium wilt
(Mandel and Baker, 1991). Induction of resistance by ASM has also been recorded in
many plants (Cole, 1999; Narusaka et al., 1999; Elmer, 2006). In a resistance inducing
experiment on eggplant against Ralstonia solanacearum, role of chitosan, salicylic acid,
methyl salicylate and methyl jasmonate elicitors on cell wall strengthening and
activation of defense enzymes were investigated. After elicitor applications, significant
increase in total phenolic substance content were observed at roots. Peroxidase activity
were found highest 24 h after CHT and SA treatments (Mandal, 2010).
Lignin deposition of infected vascular bundles was demonstrated
histochemically within papilla and nearby walls in several plants (Aist, 1983). The
detection of phenolics and lignin that accumulate during infection has been based
mainly on fluorescence microscopy and histochemistry as discussed by Nicholson and
Hammerschmidt (1992). In particular, histochemical stains have been used for
localization of induced changes in cell wall polymers, which are insoluble and thus
more difficult to quantitate by conventional means. The multifunctionality of lignins
permits them to react with many different histochemical reagents to produce coloured
products (Vance et al., 1976). Many studies agree that the accumulation of phenolics
like lignin may be associated with cell death, thus being the first step in plant defence
mechanisms in infected plants (Cohen et al., 1990). Lignin-like materials were localized
in cells undergoing HR has been implicated in highly cultivar specific resistance
expressed by wheat to the rust fungus Puccinia recondita (Southerton and Deverall,
1990). Although most of the studies on plant lignin deposition are based on fungal
pathogens, bacterial infections may also lead to lignin accumulation (Soylu, 2006).
This is the first study on induction of resistance against the wilting agent,
Fusarium oxysporum f. sp. melongenae, on eggplant. The results of the study
demonstrate that susceptible eggplants enhance a systemically induced resistance to
Fomg infection in response to ASM and FOM application. Similar observations have
highlighted the ASM as a commercial product in activating SAR in tobacco (Friedrich
et al., 1996) and tomato (Benhamou and Belanger, 1998).
In conclusion, the plant defence activator ASM seems to be a useful tool for
induced resistance studies in eggplant as observed in other plant species. Cell-wall
lignification at the reaction sites would be involved in resistance to non-pathogen
Fusarium strains. A good knowledge on the mechanisms responsible for plant defense
should be examined under commercial conditions. Further studies will be necessary to
determine the association of enhanced pathogenesis-related (PR) proteins, especially
chitinases and β-1,3-glucanases with systemic resistance.
28
H. ALTINOK
ÖZET
ACIBENZOLAR-S-METHYL VE NON-PATOJEN FUSARİUM OXYSPORUM
MELONİS (FOM) TARAFINDAN PATLICAN FİDELERİNDE
FUSARİUM SOLGUNLUK HASTALIĞINA KARŞI
SİSTEMİK DAYANIKLILIĞIN TEŞVİKİ
Bitki aktivatörü acibenzolar-S-methyl (ASM; Actigard 50 WG) ve patojenik
olmayan bir Fusarium oxysporum (FOM; Fusarium oxysporum f. sp. melonis) streyninin
patlıcan fidelerinde Fusarium oxysporum f. sp. melongenae’ya dayanıklılığı teşvik
etme yetenekleri araştırılmıştır. ASM ve FOM ile patlıcandaki ön uygulamalar hastalık
şiddetini önemli derecede düşürmüştür. Hastalık şiddetindeki en büyük düşüş,
uygulamadan 72 sa sonra patojen inokulasyonu verildiğinde elde edilmiştir. Bu aralık
kullanıldığında, inokulasyondan sonraki 21. günde, pozitif kontrol bitkilerinde hastalık
şiddeti %91.0, ASM ve FOM uygulanan bitkilerde ise sırasıyla %30.1 ve %20.1 olarak
belirlenmiştir. Mikroskopi çalışmaları, teşvik edici-patojen inokulasyonu arasındaki
süre ile hipersensitif reaksiyon gösteren ksilem hücrelerindeki lignin birikimi arasında
güçlü bir bağlantı olduğunu göstermiştir. Ksilemde yoğun lignin birikiminin
gözlenmesi, her iki uygulamanın da patlıcanda bu hastalığa karşı dayanıklılığı teşvik
ettiğini göstermektedir.
Anahtar Kelimeler: Fusarium Solgunluğu, Uyarılmış Dayanıklılık, Non-patojen
Fusarium oxysporum, ASM, Lignin, Patlıcan
ACKNOWLEDGEMENTS
The author is grateful to the late Dr. Yeter Canıhoş; Dr. M. Kamberoğlu from
Çukurova University for kind help at many stages of this work; Dr. Seral Yücel (Plant
Protection Central Research Institute, Adana) for kindly assistance; Dr. S. Soylu
(Department of Plant Protection, Mustafa Kemal University, Turkey) for use of his
laboratory facilities and technical assistance. This study was supported by the Academic
Research Projects Unit of Çukurova University and Süleyman Demirel University.
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32
ISSN 0378 - 8024
The Effect of Charcoal Rot Disease (Macrophomina phaseolina),
Irrigation and Sowing Date on Oil and Protein Content
of Some Sesame Lines
Pınar SAĞIR*
Abuzer SAĞIR**
Tahsin SÖĞÜT***
* Plant Protection Research Station, 21110 Diyarbakır, Turkey
** Department of Plant Protection, Agricultural Faculty, Dicle University, 21280 Diyarbakır, Turkey
*** Department of Crop Science, Agricultural Faculty, Dicle University, 21280 Diyarbakır,
Turkey e-mail: [email protected]
ABSTRACT
This study was conducted to determine the effect of charcoal rot disease
(Macrophomina phaseolina) on oil and protein content of some sesame lines cultivated
under different conditions at the experimental area of Dicle University Faculty of
Agriculture, in 2006 and 2007. In this study, 6 sesame lines (B–60, C–7, C–36, C–53,
Y–7 and Y–11) which are of Mediterranean and Southeastern Anatolia Region origin
and 3 isolates which belong to the M. phaseolina fungus are treated. Before sowing,
experimental area was inoculated artificially by disease factor. Sesame seeds were sown
on 05 May 2006, 22 June 2006, 11 May 2007 and 22 June 2007, respectively. The
experiment was established in a split-split plot design with three replications. At crop
maturity, unhealthy and healthy plants on each plot were harvested separately, and
adequate amount of seed samples were taken. In both years, the seeds from each plot
were taken after harvest for determining oil and protein content. Average protein and
oil content were recorded as 23.18% and 45.48%, respectively. Protein and oil contents
were affected by sowing date, irrigation and disease. According to the obtained data,
protein content of seeds obtained from unhealthy plants was found lower when
compared to the healthy plants, and oil content was found higher. The highest protein
content obtained from early sowing, dry conditions and healthy plants (25.99%), and the
lowest content from late sowing, irrigated and unhealthy plants (20.64%). The highest
oil content obtained from early sowing, irrigated and unhealthy plants (46.54%) and the
lowest oil content from early sowing, irrigated and healthy plant (44.70%).
Key words: Sesame, protein, oil, sowing date, irrigation, charcoal rot disease,
Macrophomina phaseolina
33
THE EFFECT OF CHARCOAL ROT DISEASE (Macrophomina phaseolina),
IRRIGATION AND SOWING DATE ON OIL AND PROTEIN
CONTENT OF SOME SESAME LINES
INTRODUCTION
Sesame (Sesamum indicum L.) is one of the oldest cultivated plants which are
grown all over the world and has been known since ancient ages. It has an important
role in human nutrition by virtue of rich nutrients it has contained. Sesame seeds are
used in baking cupcake, bread and cream-cake, as well as they are directly consumed as
snack food by being roasted. Furthermore, sesame seeds are used in making tahini by
way of seed crushing. Acquired product, i.e. tahini, can be directly consumed by being
blended with honey and molasses, as well as it is also used as making sesame seed paste
mixed with thick nuts and cereals (Atakişi, 1985; Arıoğlu et al., 2010). Oil at the rate of
40-60% is available in sesame seeds. Sesame oil is quite resistant to spoiling thanks to
the “sesamol and sesamolin” it has contained. Sesame oil, the sustenance of which is
high, is used as vegetable oil and margarine. As it is being used as foodstuff, sesame oil
is also used in food industry as raw material (İlisulu, 1973; Baydar, 2005).
Sesame is successfully cultivated in irrigated and dry areas in Aegean,
Mediterranean, Marmara and Southeastern Anatolia Region in Turkey. The importance
of this cultivated plant is gradually increasing by the reasons of short vegetation period,
low production cost and the possibility of its being cultivated as second crop (Arıoğlu et
al., 2010). In Turkey, sesame cultivation is being carried out in an area of 31.824 ha and
23.460 ton crops are received in total (Anonim, 2010).
Sesame seeds are highly rich in protein. Within the scope of studies carried out,
it was determined that the protein content varies between 20.0% and 25.18% (Bahkali,
1998; Ünal and Yalçın, 2008; Nzikou et al., 2009; Kanu, 2011). In addition to the
protein and oil content, sesame seeds are quite rich in carbohydrates, mineral substances
and fatty acids, too (Uzun et al, 2002; El Khier et al., 2008; Nzikou et al., 2009;
Alyemeni et al., 2011).
One of the most important factors which affect the sesame cultivation negatively
in our country and Southeastern Anatolia Region is charcoal rot disease/wilt disease. It
has been determined that Macrophomina phaseolina (Tassi) Goid., Fusarium oxysporum
f. sp. sesami (Zaprometoff) Castellani, Rhizoctonia solani Kühn., Stemphylium sp.
fungus cause this disease (Tatlı and Sağır, 1992, Ataç et al., 1994, Gürkan, 1995).
M. phaseolina fungus causes greater harm, especially in the event that host plants are
weakened by stress and dehydrated. Disease factor may infect the plants in a wide
temperature range from 20 0C to 35 0C, as based on the condition of soil water retention
(Olaya and Abawi, 1996; Diourte et al., 1995). Within a survey study carried out in
Diyarbakır and Şanlıurfa provinces in Southeastern Anatolia Region with the object of
determining fungal diseases encountered in sesame, it has been stated that the average
prevalence rate of wilt disease is 88.8%, disease rate is 8.9% and the major and most
important factor is M. phaseolina, and also stated that this fungus has been obtained at
34
P. SAĞIR, A. SAĞIR, T. SÖĞÜT
the rate of 65.62% in isolations made (Gürkan 1995). This pathogen causes wilting of
growing plants completely by forming necrosis in root collar of the plant.
This study was carried out to determine the effect of charcoal rot disease
(Macrophomina phaseolina) on protein and oil content of seeds gathered from sesame
lines cultivated under different conditions.
Some sesame lines which are of Mediterranean and Southeastern Anatolia
Region origin were cultivated in the early and late period and under the irrigated and
dry conditions, in Diyarbakır province. Experiments established in order to determine
the effect of charcoal rot disease which is caused by Macrophomina phaseolina fungus
on the protein and oil content of sesame seeds.
In 2006-2007 growing seasons, experiments were established in a field of the
Research Area of Dicle University Faculty of Agriculture where is naturally
contaminated by the disease factor (Macrophomina phaseolina) and the disease has
been encountered in the previous years. In the study, 6 different sesame lines which are
of Mediterranean origin (Y–7 and Y-11) and of Southeastern Anatolia Region origin
(B-60,C-7, C-36 and C-53) were used as material.
The field experiments were laid out in a split-split plot design with three
replications. Plots were 4 m long and 1.4 m wide with two rows spaced by 0.7 x 0.15 m.
In order that the occurrence of disease can be seen intensely, the soil was
artificially inoculated by the disease factor M. phaseolina after it has been made ready
for sowing later than the cultivation. Therefore, 3 M. phaseolina isolates previously
isolated were left for incubation for a time period to last 15 days at 22 oC, after they
were placed in sterilized petri and erlenmeyers that contained wheat-medium (1000 g
wheat + 800 ml water). Afterwards, growing inoculum was broken into pieces and
applied into 1 m2 soil as to be 75 g on the date of 10 April 2006. As to make soil
inoculation easily and homogeneously, after this inoculum was blended with sand at the
rate of 1/5 (inoculum in 1 proportion + stream sand in 5 proportions), it was applied into
the soil on each parcel evenly. Following the inoculation, the soil was tillaged by
rotovator as to be 5-10 cm in depth and the homogeneous blending of inoculum with
soil has realized (Sağır et al., 2009).
Before sowing, basal fertilizer was applied into the experimental plots in the
dose of 100 N kg ha-1 and 100 P2O5 kg ha-1 as 20-20-0. Sesame seeds were manually
sown to the lines which are opened on experimental plots at two different planting dates
within the years 2006 and 2007 [(Dates for the 1st sowing time (early sowing) are
35
05.05.2006 and 11.05.2007; dates for the 2nd sowing time (late sowing) are 22.06.2006
and 22.06.2007)]. At the second sowing time, sowing was performed after soil was
irrigated. After the emergence of plants and in the period when 3-4 actual leaves exist,
thinning was performed in the way that 0.15 m intra-row spacing.
Usual agricultural procedures were maintained throughout the season and weed
control was mechanically carried out. With the aim of weed control and soil ventilation,
3 hoeing treatments were performed. In case of need, plants on irrigated plots were
provided with irrigation water, as 5 times in 2006 and 4 times in 2007, by means of
furrow irrigation method.
Following the maturation of plants at the end of the growing season, healthy and
unhealthy plants on each plot were harvested separately and laid on a nylon cover. After
plants have completely dried out, seeds were separated from the plants by being shaken.
After seeds in adequate amount (20 grams) had been received for each plot and
milled properly, they were put into nylon bags by being labeled and preserved in
deepfreeze at -18 0C. Before carrying out protein and oil analyses, seed samples were
kept in Pasteur’s furnace at 70 0C for approximately two hours and by this means,
dehumidification of excessive humidity has been ensured. In order to determine the
protein and oil contents, a 25 g sample of dry seeds from each plot were finely
grounded. The each sample was analyzed for crude protein content with a model LECO
FP-528 analyzer (LECO Corp., Joseph, MI), three reading for protein was taken from
three sub-samples and their average value was recorded. The crude protein content in
seeds was estimated by applying the factor N x 6.25 to the seed N content. Sesame flour
was extracted into petroleum ether using soxhlet apparatus for 4h as per process of the
instrument (AOAC, 1960). Oil contents were determined by weight differences. All
values are mean of observations in three independent samples. Seed protein and oil
contents were expressed in % on a dry matter basis.
The data were analysed by a standard procedure for analysis of variance, and the
significance of differences among sample means was determined by LSD test using
SPSS 17.0. In this way, the effect of sowing date, irrigation and charcoal rot disease in
sesame which was caused by Macrophomina phaseolina fungus on protein and oil
content were determined.
Average protein and oil contents of sesame lines which were cultivated under
different conditions were shown in Table 1. As it seems in Table 1, the protein content
of sesame lines ranged from 22.55% to 23.61%, and oil content of them varied between
44.85% and 46.90%. The highest protein and oil content was obtained respectively from
36
the lines of Y-7 and C-7. Bahkali et al. (1998) reported that the protein content of the
seeds of white hulled and dark hulled sesame of which different geographical origins
varies between 23.13% and 25.18% and the oil content of them ranges from 47.02% to
49.07%. Ünal and Yalçın (2008) determined that the average protein content of seed
samples taken from 4 different sesame types (Gölmarmara, Özberk, Muganlı, Çamdibi)
cultivated in different regions of Turkey is 21.00% and the oil content is 54.26%, and
Baydar et al.(1999) determined that the oil contents of 16 fine lines picked out of 160
sesame lines varies between 57.2% and 63.25%.
Table 1. Average protein and oil content of sesame lines (%)
Lines
Protein Content (%)
Oil Content (%)
1(B-60)
23.31a
45.18b
2(C-7)
23.14a
46.90a
3(C-36)
22.55b
45.64b
4(C-53)
23.29a
44.85b
5(Y-7)
23.61a
45.36b
6(Y-11)
23.15a
44.96b
Average
23.18
45.48
*Values within a column followed by different letters differ significantly (P< 0.05) according to LSD test.
According to the findings obtained, protein and oil contents of sesame lines
varied statistically by the years, sowing time, irrigation and status of disease (Table 2).
When all these factors are taken into consideration, the average protein content of six
sesame lines found as 23.18% and the oil content as 45.48%. Just as in the previous
studies carried out in this respect, it was detected that the protein and oil contents of
sesame seeds vary by the color of seed hull, growing regions, the point of which capsule
exists on the plant and sowing date (Mosjidis and Yermanos., 1984; Bahkali et al.,
1998; Baydar and Turgut, 1994; Ünal and Yalçın, 2008; Kanu, 2011).
In early and late sowings, it was found that the protein content of sesame seeds
received from unhealthy plants cultivated under irrigated and dry farming conditions
was lower in comparison with the ones received from healthy plants and the oil content
was higher. It was determined that the average protein content of unhealthy plants is
20.64-24.86% and the oil content of them was 44.85-46.54%, and respectively, the
protein and oil contents of healthy plants were 21.31-25.99% and 45.61-44.70% (Table
3). Any source or reference indicating the effect of Macrophomina phaseolina fungus
on oil and protein content of sesame seed could not be reached.
Although the sowing time, irrigation and the interaction between disease and
lines could not be found statistically significant, the highest average protein content has
37
been obtained from early sowing, dry and healthy plants (25.99%) and the lowest
average protein content from late sowing, irrigated and unhealthy plants (20.64%). The
highest average oil content was obtained from early sowing, irrigated and unhealthy
plants (46.54%), and the lowest average oil content was obtained from early sowing,
irrigated and healthy plant seeds (44.70%) (Table 3).
As a consequence, it was determined that sowing time, irrigation conditions,
sesame type/line and disease status had an effect on oil and protein contents of sesame
seeds. It will be useful to carry out studies especially in respect of disease.
Table 2. Protein and oil content (%) of sesame lines as affected by years, sowing time, irrigation and
charcoal rot disease.
Factors
Years
Sowing Time
Irrigation
Disease
Average
Characters
Protein Content (%)
Oil Content (%)
2006
23.48a
44.80b
2007
22.87b
46.09a
Early
23.92a
45.25a
Late
22.43b
45.71a
Irrigated
21.69b
45.75a
Dry
24.66a
45.21b
Unhealthy
22.68b
45.89a
Healthy
23.67a
45.07b
23.18
45.48
*Values within a column followed by different letters differ significantly (P< 0.05) according to LSD test.
38
Table 3. Mean values of protein and oil contents (%) of sesame lines as affected by sowing time, irrigation
and charcoal rot disease.
Sowing
Time
Irrigation
Disease
Unhealthy
Irrigated
Healthy
Early
Unhealthy
Dry
Healthy
Unhealthy
Irrigated
Healthy
Late
Unhealthy
Dry
Healthy
Lines
Protein
(%)
1(B-60)
2(C-7)
3(C-36)
4(C-53)
5(Y-7)
6(Y-11)
1(B-60)
2(C-7)
3(C-36)
4(C-53)
5(Y-7)
6(Y-11)
1(B-60)
2(C-7)
3(C-36)
4(C-53)
5(Y-7)
6(Y-11)
1(B-60)
2(C-7)
3(C-36)
4(C-53)
5(Y-7)
6(Y-11)
1(B-60)
2(C-7)
3(C-36)
4(C-53)
5(Y-7)
6(Y-11)
1(B-60)
2(C-7)
3(C-36)
4(C-53)
5(Y-7)
6(Y-11)
1(B-60)
2(C-7)
3(C-36)
4(C-53)
5(Y-7)
6(Y-11)
1(B-60)
2(C-7)
3(C-36)
4(C-53)
5(Y-7)
6(Y-11)
21.90
20.76
20.81
21.58
23.06
21.81
23.30
22.96
22.83
23.76
23.00
23.15
24.65
24.85
23.88
25.38
25.43
25.05
26.26
25.75
25.71
26.21
26.30
25.71
20.68
20.46
20.60
20.01
20.60
21.51
21.66
21.76
20.80
21.66
21.53
20.45
23.70
23.83
21.85
23.80
24.61
23.60
24.38
24.73
23.96
23.95
24.40
23.91
Oil (%)
47.13
49.93
48.28
43.16
45.31
45.45
46.00
44.51
43.11
43.31
45.71
45.58
45.00
45.50
46.25
45.53
43.73
43.60
44.93
47.08
44.85
44.20
43.65
44.26
45.73
47.76
42.41
43.31
49.28
48.30
46.91
45.45
48.90
44.51
45.33
42.55
42.68
48.81
43.93
47.78
46.73
45.78
43.05
46.15
47.36
46.98
43.13
44.21
Average
Protein
(%)
Average
Oil (%)
21.65
46.54
23.16
44.70
24.86
44.85
25.99
44.83
20.64
46.13
21.31
45.61
23.55
45.95
24.22
45.15
39
ÖZET
KÖKBOĞAZI ÇÜRÜKLÜĞÜ (Macrophomina phaseolina ), SULAMA VE
EKİM ZAMANININ BAZI SUSAM HATLARININ
YAĞ VE PROTEİN İÇERİKLERİ ÜZERİNE ETKİSİ
Bu çalışma, kökboğazı çürüklüğü hastalığı (Macrophomina phaseolina)’ ın farklı
koşullarda yetiştirilen bazı susam hatlarının protein ve yağ içeriklerine olan etkisini
belirlemek amacıyla, 2006-2007 yıllarında Dicle Üniversitesi Ziraat Fakültesi Araştırma
alanında yapılmıştır.
Çalışmada, Akdeniz ve Güneydoğu Anadolu Bölgesi kökenli 6 susam hattı (B–
60, C–7, C–36, C–53, Y–7, Y–11) ile M. phaseolina fungusuna ait 3 izolat
kullanılmıştır. Ekimden önce deneme alanı hastalık etmeni ile yapay olarak inokule
edilmiştir. Susam tohumları, 05.05.2006, 22.06.2006, 11.05.2007 ve 22.06.2007
tarihlerinde ekilmiştir. Denemeler, bölünen bölünmüş parseller deneme desenine göre,
üç tekerrürlü olarak kurulmuştur.
Mevsim sonunda bitkilerin olgunlaşmasından sonra, her parseldeki hasta ve
sağlam bitkiler ayrı ayrı hasat edilerek yeteri miktarda tohum örnekleri alınmıştır.
Tohum örnekleri değirmende öğütüldükten sonra dipfirizde muhafaza edilmiştir. Protein
analizi için her örnekten 0.25 g alınarak LECO-FP-528 cihazında (LECO Corp, Joseph,
MI) analiz edilerek ham azot (% N) bulunmuştur. Elde edilen % N oranları 6.25 faktörü
ile çarpılarak (% N x 6.25) tohum protein oranları belirlenmiştir. Yağ analizleri için
aynı şekilde hazırlanan örneklerden 5 g alınarak Soxhlet cihazında 70 0C sıcaklıkta
organik çözücü (dimethylether) ile ekstraksiyon yöntemine göre analiz edilmiş ve elde
edilen değerler % ‘de olarak hesaplanmıştır.
Susam hatlarının ortalama protein oranı %23.18, yağ oranı ise % 45.48 olarak
bulunmuştur. Protein ve yağ oranları, ekim zamanı, sulama koşulları ve hastalık
durumuna göre farklılık göstermiştir. Elde edilen verilere göre, hastalıklı bitkilerden
elde edilen tohumların protein içeriği, sağlıklı bitkilere göre daha düşük yağ oranları ise
daha yüksek bulunmuştur. En yüksek protein oranı; erken ekim, susuz ve sağlam
bitkilerden (%25.99), en düşük ise geç ekim, sulu ve hasta bitkilerden (%20.64) elde
edilmiştir. En yüksek yağ oranı; erken ekim, sulu ve hasta bitkilerden (%46.54), en
düşük yağ oranı ise; erken ekim, sulu ve sağlam bitki tohumlarından (%44.70) elde
edilmiştir.
Anahtar Kelimeler: Susam, protein, yağ, ekim zamanı, sulama, kökboğazı
çürüklüğü, Macrophomina phaseolina
40
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42
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TURKISH PHYTOPATHOLOGICAL SOCIETY EDITORIAL BOARD

Transkript

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