Current Status of Tilletia Species in Vojvodina, Serbia

Current Status of Tilletia Species in Vojvodina, Serbia
Vesna Župunski, Radivoje Jevtić
Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia
e-mail: [email protected]
Abstract
Analysis of 151 samples of basic, certified and commercial non-processed seed of wheat in
Vojvodina revealed that 129 samples were contaminated with species of bunt pathogens in the genus
Tilletia. The predominant species was T. caries, which was found in 127 samples and is known to occur
in Serbia. However, teliospores of Tilletia species which have not been registered in Serbia before
were also found in 12 samples. These teliospores displayed either a prominent gelatinous sheath with
conspicuous depth of reticulations or a very small-diameter areolae. It was assumed that some of the
teliospores with prominent gelatinous sheath belong to the quarantined smut species T. contraversa,
while the others were identified based on morphological traits as T. bromi. Teliospores which were
distinct from those of T. caries due to smaller-diameter areolae were assumed to belong to T. fusca
var. fusca. Using rep-PCR fingerprinting T. caries, T. laevis and T. contraversa were distinguished,
thus rep-PCR fingerprinting has been considered as a useful tool for making distinction between
Tilletia species. The contamination level of four of 16 commercial seed samples exceeded threshold
contamination levels of 900 teliospores per seed. On average, basic and certified seed samples were
contaminated with about 1 teliospore per 10 seeds.
Key words: T. caries, T. contraversa, T. bromi, wheat, rep-PCR fingereprinitng
Introduction
Bunt of wheat is a fungal disease that occurs worldwide and is caused by Tilletia species. Additional
hosts of Tilletia include rye, barley, triticale and many other species of the family Poaceae (Goates,
1996). The most common Tilletia species in Serbia are T. caries (DC.) Tul. and T. foetida (Wall.) Liro
(Jevtic et al., 1997). Tilletia caries and T. foetida are causal agents of common bunt. A putative hybrid of
T. caries and T. foetida, called Tilletia triticoides Savalescu and Tilletia intermedia Grassner, was reported
on wheat in the north-eastern part of the country by Kostić and Smiljković (1968). Quarantine species
for Serbia include both T. contraversa Kühn (Jevtić R., 2004), which incites dwarf bunt, and T. indica
Mitra, which causes Karnal bunt and is listed as an A1 quarantine pest for the EPPO region (European
and Mediterranean Plant Protection Organization). Neither of them have been reported in Serbia.
The first report of Tilletia species in Serbia was made at the end of the 19th century (Simić, 1895).
In the middle of the 20th century, the most commonly registered species was T. foetida (Minev, 1951),
but in the 1990s the predominant species was T. caries (Jevtić et al., 1997a; Jevtić et al., 1997b). In the
1960s common bunt was successfully controlled (Jevtić et al., 1997, Delalić et al., 2005), but in the
1990s, because of the economic embargo imposed on our country, common bunt had a severe impact
on wheat seed production. Changes in wheat production during the latter period, including sowing
undeclared seed, discontinuation of fungicide treatments, appearance of new races, and prevalence
of disease-conducive environment, contributed to the outbreak of common bunt (Jevtić et al., 1997a;
Jevtić et al., 1997b; Jevtić et al., 2010; Jevtić et al., 2012). In addition, Koprivica et al. (2009) investigated
the effects of Tilletia spp. inoculum source and environmental conditions on the incidence of wheat
spike infection. In that study, wheat seeds were artificially contaminated with Tilletia spp. teliospores
176
originating from the municipality of Novi Sad (South Bačka District, Autonomous Province of
Vojvodina); it was observed that seedborne inoculum caused spike infection during the third and
fifth 10-day period after sowing. Since teliospores of T. caries and T. foetida infect wheat during
germination, which takes place in the first 11 days after sowing, these later-season infections were
assumed to be caused by either hybridization between T. caries and T. contraversa or a new race of
T. caries. Monitoring the presence of Tilletia species, varieties and races in wheat production areas
is very important as well as breeding for resistance to these pathogens (Matanguihan et al., 2011;
Jevtić et al., 1998; Jerković et al., 2005), since European agriculture has been moving toward organic
production and low-input farming systems with reduced chemical inputs in crop production
(Matanguihan et al., 2011).
In addition, it is essential to monitor the contamination level of organically produced wheat seed
in order to determine whether the regulated spore threshold is exceeded. The currently prescribed
spore thresholds for common bunt in organic agriculture vary in different countries; they include one
spore/seed in Scotland and in the United Kingdom, 10 spores/seed in Austria and Switzerland, and
20 spores/seed in Germany. In Denmark, intervention is recommended at the first detection of spores
in seed sample (Matanguihan et al., 2011). Contamination limits for T. contraversa in Switzerland,
Austria and Scotland are the same as the previously mentioned (Micheloni et al., 2007). In Serbia,
the allowed level of Tilletia spp. contamination, determined by the Ministry of Agriculture, Trade,
Forestry and Water Management, for conventionally produced basic and certified seed of wheat is the
same: 0%. The allowed level of Tilleta spp. contamination for conventionally produced commercial
seed is 0.01% (1 bunted seed per 10,000 seeds).
Monitoring the presence of T. caries and T. contraversa in wheat seed samples is very difficult when
it is based only on morphological characteristics of teliospores (Goates, 1996). Tilletia controversa and
T. bromi which is causal agent of grass bunt could have teliospores with overlapping morphological
characteristics, too (Pimentel et al., 2000; Boyd and Carris, 1998; Boyd et al., 1998; Goates, 1996;
Castlebury and Farr, 2002).
Molecular techniques have earned a significant role in identification of different plant and pathogen
genotypes (Kondić-Špika et al., 2009; Koprivica et al., 2009a). Furthermore, they are sometimes the
only reliable methods for identification of certain plant pathogens, including species of the genus
Tilletia. McDonald et al. (2000) found rep-PCR fingerprinting technique to be very useful for
distinguishing T. indica from T. walkeri, as well as T. contraversa from T. caries and T. bromi. Župunski
et al. (2011) obtained the same results as McDonald et al. (2000) concerning coefficients of similarity
among Tilletia species. Even though it has not been confirmed that selective primers which can
differentiate T. contraversa from T. caries can also make distinction between T. contraversa and the
morphologically similar T. bromi (Yuan et al., 2009; Liu et al., 2009; Gao et al., 2010; Gao et al., 2011),
rep-PCR fingerprinting is nevertheless considered to be the most precise technique for distinguishing
Tilletia species found in wheat seed samples.
Materials and methods
Collection of wheat seed samples. One hundred and fifty one non-processed seed samples
of autumn-sown wheat were collected in cooperation with regional phytosanitary laboratory
Agroinstitut, Sombor during the 2007-2008 harvest season. Out of 151 non-processed seed samples,
135 were categorized as basic and certified seed, whereas 16 were categorized as commercial seed.
Basic, certified and commercial seed categories are defined by OECD - Organization for Economic
Co-operation and Development (2008). Seed samples were collected from 39 municipalities in
the Autonomous Province of Vojvodina (northern part of Serbia). Wheat grain was sampled from
trucks in order to produce 1 to 2 kg mixed seed samples. Seed samples were stored at 15°C with
177
Current Status of Tilletia Species in Vojvodina, Serbia
relative humidity <60% (Elias et al., 2007). Teliospores were extracted from 50 g subsamples (OEPP/
EPPO, 2007).
Teliospore extraction and identification on the basis of teliospore morphology. Teliospore
extraction and morphological characterization were carried out in the Laboratory of Phytopathology
(Maize Research Institute “Zemun Polje”, Belgrade). By using the size-selective sieving wash method
(Peterson et al., 2000) and the OEPP/EPPO diagnostic protocol for Tilletia indica (2007), teliospores
were extracted from 50 g subsamples of each grain sample. These protocols were modified by using
a 10 μm mesh nylon sieve instead of a 20 μm nylon sieve. Extracted teliospores were mounted in
15% glycerol and examined by light microscopy at 250x magnification. Teliospore diameter, depth of
reticulation, number of meshes per teliospore diameter, areola diameter and thickness of gelatinous
sheath were measured at 1000x magnification. If teliospores were not found in the first 50 g subsample,
then two additional 50 g subsamples were examined in order to determine the presence of Tilletia
species teliospore with confidence level of 99% (OEPP/EPPO, 2007).
Quantification of contamination level. Contamination level per sample was determined by
calculating total number of teliospores per 50 g subsample using of the Breed Method. Teliospore
suspensions were obtained after washing 50 g seed subsample with the use of size-selective sieving
wash method. Pelleted teliospores were suspended with 100 µl (or more) of 15% glycerol, depending
on the pellet volume (OEPP/EPPO, 2007). In order to estimate the total number of teliospores per 50 g
subsample, it was necessary to determine the average number of teliospores per field of view, the total
number of fields of view per cover slip area, and the total number of cover slip areas needed to exam
the entire teliospore suspension.
Method for teliospores germination and mycelial mat production. Teliospores were extracted
from both the microscope slide and the cover slip by using 10 μm mesh nylon sieve instead of 20
μm nylon sieve and surface-sterilized with 0.2% NaOCl. In order to initiate teliospore germination
and production of primary spores of T. caries, T. foetida and T. contraversa, one part of the teliospore
suspension was plated on 2% water agar with antibiotics (AWA: 2% Technical agar No. 3, Oxoid; 60
mg penicillin-G (Na salt) and 200 mg streptomycin sulphate per L of agar) and incubated for 10-15
days at 16°C whereas the other part of suspension, which was also plated on 2% AWA, was incubated
for 3-6 weeks at 5°C in the presence of light (Goates, 1996). Small blocks of agar bearing germinated
teliospores or colonies were cultivated in potato dextrose broth (PDB) for 15-20 days and incubated at
16-20°C in the dark, in order to produce at least 100 mg fresh weight of material, which was afterwards
frozen and maintained at -80°C.
Isolation of DNA and rep-PCR amplification. DNA was isolated from mycelial mats using the
procedure of Möller et al. (1992). Frozen material, obtained from 100–500 mg of fresh weighted
material, was placed in a microtube and ground with gradual addition of liquid nitrogen. Proteinase
K, 10% cetyltrimethylammoniumbromid (CTAB) and SEVAG (chloroform: isoamylalcohol, 24:1, v/v)
were used for DNA isolation. DNA was precipitated with isopropanol. Isolated DNA was resuspended
in 0,1x TRIS-EDTA buffer (TE).
Identification of Tilletia species using rep-PCR fingerprinting was carried out in the Laboratory
for Biotechnology. Rep-PCR assays were performed with 19 isolates of Tilletia species; 15 isolates
originated from teliospores which were extracted from nine non-processed 50 g seed subsamples,
whereas four isolates were positive controls for T. contraversa, T. caries, T. foetida and T. walkeri (Table
1). Isolates used as positive controls for T. contraversa, T. foetida and T. walkeri were obtained from
Centraalbureau voor Schimmelcultures (CBS) collection, while isolate used as positive control for T.
caries was obtained from Institute of Field and Vegetable Crops, Novi Sad (Table 1).
178
Table 1. DNA samples and sources of each sample.
Sample
number
Sources
Supplier
Locality
GPS Coordinate
Latitude
Longitude
Melenci
N 45 31.225
E 20 18.947
Srpska
Crnja
N 45 43.579
E 20 41.925
Melenci
N 45 31.225
E 20 18.947
Bačka
Palanka
N 45 15.196
E 19 23.977
Apatin
N 45 40.140
E 18 59.063
1
positive control for
Tilletia walkeri
CBS
(accession number:
121956)
USA
2
positive control for
Tilletia laevis
CBS
(accession number:
121950)
Iran
3
positiv control for
Tilletia controversa
CBS
(accession number:
121952)
Unknown
4
mercantile wheat 1.1
5
mercantile wheat 1.2
6
mercantile wheat 1.3
7
mercantile wheat 4.1
8
mercantile wheat 4.2
9
mercantile wheat 2.1
10
mercantile wheat 2.2
11
mercantile wheat 11.1
12
mercantile wheat 11.2
13
cultivar Ljiljana 1
14
cultivar Ljiljana 2
15
mercantile wheat 6
Mokrin
N 45 56.367
E 20 24.467
16
mercantile wheat 7
Kikinda
N 45 50.072
E 20 27.825
17
positive control for
Tilletia tritici
Novi Sad
N 45 15.710
E 19 51.179
18
artificially contaminated Institute of Field and
wheat seed with T. caries Vegetable Crops, Novi Sad
19
mercantile wheat 14
Inđija
N 45 03.167
E 20 05.042
Agroinstitut, Sombor
Institute of Field and
Vegetable Crops, Novi Sad
Agroinstitut, Sombor
The rep-PCR protocol was performed using modified procedures described by McDonald et al.
(2000) and the OEPP/EPPO diagnostic protocol for T. indica (2007). Rep-PCR fingerprints were
obtained with the use of the following primers: REP 1R (5’-IIIICGICGICATCIGGC-3’) and 2I
(5’-ICGI CTTATCIGGCCTAC-3’); ERIC 1R (5’-ATGTAAGCTCCTGGGGATTCAC-3’) and 2I
(5’-AAGTAAGTGACTGGG GTGAGCG-3’) and BOX 1A1R (5’-CTACGGCAA GGC GAC GCT
179
Current Status of Tilletia Species in Vojvodina, Serbia
GAC G –3’). Briefly, 100 ng of purified DNA was used as template in a 50 μl reaction mixture containing
1x PCR buffer, 0.5 μM of each primer, 0.2 mM of each deoxynucleotide triphosphates, 6.25 units of Taq
DNA polymerase, and 1.5 mM of MgCl2. PCR cycling parameters were: an initial denaturation for 7
min at 95°C; 35 cycles consisting of 94°C for 3 s, 92°C for 30 s, then either 40°C (REP primers) or 50°C
(ERIC/BOX primers) for 1 min; extension for 8 min at 65°C; and a single final extension for 8 min at
65°C, followed by cooling at 4°C. DNA fragments in 9 μl of the amplified PCR product were separated by
electrophoresis on 1.5% agarose gels at 4°C in Tris- acetate- EDTA buffer (TAE; 0.04 M Tris-acetate and 1
mM EDTA, pH 8.0) at 2 V/cm for 18 to 19 h. Twenty-line gels were normalized using 1-kb DNA ladders
(Fermentas) loaded on both sides and in the center of the gels. DNA bands were stained with ethidium
bromide, visualized under a UV transilluminator, and photographed with a digital camera. Positions of
faint and strong DNA fragments on agarose gel were scored visually (McDonald et al., 2000). A matrix
of binary variables - band present (1) or band absent (0) - was generated for each genotype. A similarity
coefficient defined by Jaccard (1908) was used to estimate genetic similarity among isolates. Cluster
analysis was carried out on the matrix of genetic similarity using the unweighted pair group method
with arithmetic averages (UPGMA). Using NTSYS-pc software (Rohlf, 2000), binary data including
coefficients of similarities and UPGMA clustering were computed.
Results and discussion
Incidence of Tilletia species in non-processed seed of wheat in Vojvodina. Župunski et al. (2012)
revealed that 129 out of 151 non-processed seed samples of wheat from Vojvodina were contaminated
with teliospores of Tilletia species. Tilletia species were found in all categories of winter wheat samples
including basic, certified and commercial seed. The predominant species was T. caries which was
found in 127 samples. In addition to T. caries, T. foetida and Tilletia spp. were found in 11 and 12
samples, respectively.
Teliospores of some Tilletia spp., which were found in five samples (3.3% incidence among samples),
were morphologically distinct from those of T. caries due to a clearly differentiated gelatinous sheath
and depth of reticulations (Fig. 1). Teliospores were 18-24 μm in diameter. It was assumed that these
anomalous Tilletia spp. teliospores belong to either T. contraversa or T. bromi on the basis of gelatinous
sheath thickness and depth of reticulation which exceeded 1.5 µm (Župunski et al., 2012).
Figure 1. T
illetia spp. teliospore with prominent gelatinous sheath and conspicuous depth of reticulation
Scale bar = 10 μm
In seven samples, T. caries was found together with Tilletia spp. whose teliospores were
morphologically distinct from those of T. caries due to smaller diameter (≤ 1.5 μm) of areolae.
Moreover, the walls surrounding the areolae were thicker than those of T. caries; thus, areolae of all
examined teliospores lost their hexagonal shape and became almost round. Teliospores were 18 to 22
μm in diameter. It was assumed that these teliospores belong to T. fusca var. fusca after comparing
measurement data with those reported by Boyd and Carris (1998). This assumption is also supported
180
by the fact that the plant species Vulpia myuros and Vulpia bromoides, which are known to occur in
Serbia (PGR Forum Crop Wild Relative Catalogue for Europe and the Mediterranean), could be a host
for T. fusca var. fusca (Boyd and Carris, 1998).
Predominance of T. caries over the T. foetida was in line with results obtained by Stojanović et al.
(1993) and Jevtić et al. (1997). However, the first report on the presence of Tilletia spp. with a prominent
gelatinous sheath, similar to quarantine species T. contraversa, was made by Župunski et al. (2012).
Identification of Tilletia species is difficult when based solely on morphological characteristics of
teliospores. Although the presence or absence of a prominent sheath and deep reticulations are the
best characteristics for making distinctions between T. contraversa and T. caries, there is an overlap
of about 10% in the morphology of Tilletia species with reticulate exosporium. As a result, individual
teliospores of T. contraversa with sheath thickness of 1.5 μm and short reticulations can be wrongly
identified as T. caries (Goates, 1996). Liang et al. (1982) tried to determine T. contraversa and T. caries
on the bases of thickness of gelatinous sheath, depth of reticulations and germination tests, but had a
success rate of only 70%.
The similarity in morphology between T. contraversa and T. bromi is another problem for
distinguishing T. contraversa (Mathre, 1996). It is impossible to distinguish T. contraversa from T.
bromi on the basis of individual teliospore characteristics, because of overlapping of measurement
data (Pimentel et al., 2000; Boyd and Carris, 1998; Boyd et al., 1998; Goates, 1996; Castlebury and Farr,
2002). Peterson et al. (2009) analyzed presence of T. contraversa in samples of wheat export shipments
and pointed out that identification of T. contraversa and T. bromi on the basis of general morphology
and depth of reticulations (if ≥0.95 μm = T. contraversa) does not differentiate these two species; thus
there is the risk of possibly misidentifying grass bunt, leading to the risk for erroneous commodity
rejection. The hosts of grass bunts (Bromus and Festuca) can be found in and around wheat fields;
therefore, T. bromi is commonly present in wheat export shipments but in low numbers.
Contamination level of Tilletia species teliospores found in conventionally produced nonprocessed wheat seed. Contamination level by Tilletia species teliospores found in conventionally
produced non-processed wheat seed is presented in Table 2. Four of the 16 commercial seed samples
were contaminated above the threshold level of 0.01% (1 bunted seed per 10,000 commercial seed)
determined by the Ministry of Agriculture, Trade, Forestry and Water Management. Bunted seeds
were not found in seed samples. Out of 151 samples, 135 were categorized as basic and certified seed,
of which 113 were contaminated with less than 100 teliospores per 50 g subsample (< 0.1 teliospore
per seed) whereas 22 were not contaminated, with confidence level of 99% (Župunski et al., 2012).
Table 2. Contamination level of Tilletia species teliospores found in conventionally produced non-processed
wheat seed (Župunski et al., 2012).
Category of
wheat seed
Basic
Number of
teliospores per 50g
subsample
< 100
0a
Number of
Allowed contamination
Number of Total number
teliospores per
level (percent of bunted
samples
of samples
seed
seed)
≤0.1
13
14
a
0
1
0%
≤0.1
100
121
0a
21
< 1,000
<1
0a
Certified
Commercial
< 100
7
from 1,000 to 300,000 from 1 to 300
5
> 900,000
4
>900
Total
a
b
16
0,01%b
151
Non-contaminated seed samples with confidence level of 99%
One bunted seed per 10 000 seeds means 600 to 900 teliospores per seed (Josifović, 1948)
181
Current Status of Tilletia Species in Vojvodina, Serbia
It could be hypothesized that contamination of seed samples occurred during harvesting or via
contaminated storage equipment. This assumption is in accordance with assumption given by Cockerell
and Rennie (1996) who found T. caries in 60% of basic and certified seed samples. Mathre and Johnson
(1976) pointed out that in Montana low contamination level of common bunt was found even where
seed treatments are commonly used. Progress in breeding for grain yield and bunt resistance in lowpesticide-input farming systems depends on the genotypes as well as on environmental conditions
(Mladenov et al., 2011; Matanguihan et al., 2011). As a result, it is important to monitor the presence of
Tilletia species, varieties and races in wheat production areas in order to prevent resistance breakdown
as well as to facilitate sustainable organic production (Jevtić, 1998).
Teliospores recovered after microscopic examination, mycelial mat production and identification of
Tilletia species by rep-PCR. According to the OEPP/EPPO diagnostic protocol for Tilletia indica (2007),
teliospores of Tilletia species should be recovered after microscopic examination by using size-selective
sieving wash method (Peterson et al., 2000) in preparation for identification by molecular techniques.
Although Peterson et al. (2000) confirmed sensitivity of this method, Župunski et al. (2012) emphasized
a problem associated with production of mycelial mats for DNA isolation when seed samples were
contaminated with very few teliospores, such as one teliospore per 10 seeds (Table 2). On the other
hand, mycelial mats were successfully obtained from nine seed samples which were contaminated
with minimum of 1,000 teliospores of Tilletia species per 50 g seed subsample, i.e. a minimum of one
teliospore per seed (Table 2). Rep-PCR fingerprinting was applied only on the latter samples and results
were reported by Župunski et al. (2011, 2012). By using REP, ERIC and BOX primers, Župunski et al.
(2011) and McDonald et al. (2000) obtained DNA bands ranging from 100 to 4,000 bp (Fig. 2). T. caries,
T. contraversa and T. foetida do not cluster together despite a similarity of 70% (Fig. 3). In addition,
McDonald et al. (2000) and Župunski et al. (2011) reported a similarity coefficient of >80% between
isolates of T. caries (Fig. 3). According to Župunski et al. (2011), T. walkeri, T. caries, T. contraversa
and T. foetida share a similarity of about 10% (Fig. 3), which is also consistent with results obtained by
McDonald et al. (2000). Župunski et al (2011) did not use positive control for T. bromi, but it was assumed
that isolate 14 belonged to T. bromi taking into account the fact that isolates of T. bromi, T. contraversa,
T. caries and T. foetida have coefficient of similarity greater than 55% (McDonald et al., 2000). Bearing
in mind the consistency between results obtained by McDonald et al. (2000) and Župunski et al. (2011),
rep-PCR fingerprinting technique has a potential as a diagnostic method for these species.
Mc Donald et al. (2000) revealed that T. fusca var. fusca isolates on V. microstachys and V.
octoflora clustered separately and had very low similarity (20-35%). Consequently, reexamination
of the taxonomic classification of Tilletia species is highly recommended. It is clear that rep-PCR
fingerprinting can be a useful tool not only for monitoring the presence of Tilletia species and varieties
in wheat production areas but also for phylogenic and taxonomic investigations.
Figure 2. DNA fingerprints of 19 Tilletia genotypes obtained by rep-PCR technique (Župunski et al., 2011)
(a)REP-PCR, (b) ERIC-PCR, (c) BOX-PCR. Lane 1=positive control for Tilletia walkeri; Lane 2=positive
control for Tilletia foetida; Lane 3=positive control for Tilletia contraversa; Lane 4=mercantile wheat 1.1; Lane
5= mercantile wheat 1.2; Lane 6=mercantile wheat 1.3; Lane 7=mercantile wheat 4.1; Lane 8=mercantile
182
wheat 4.2; Lane 9= mercantile 2.1; Lane 10=mercantile wheat 2.2; Lane 11=mercantile wheat 11.1; Lane
12=mercantile wheat 11.2; Lane 13=cultivar Ljiljana 1; Lane 14=cultivar Ljiljana 2; Lane 15=mercantile wheat
6; Lane 16=mercantile wheat 7; Lane 17 = positive control for Tilletia caries; Lane 18= artificially contaminated
wheat seed with Tilletia caries; Lane 19=mercantile wheat 14; Lane M= DNA molecular size markers (1 kbp
ladder); sizes in base pairs.
Figure 3. Dendrogram of 19 Tilletia genotypes derived from UPGMA analysis (Župunski et al., 2011)
TW=positive control for Tilletia walkeri; TL=positive control for Tilletia foetida; TC=positive control
for Tilletia contraversa; TT = positive control for Tilletia caries; 5= mercantile wheat 1.2; 6=mercantile
wheat 1.3; 7=mercantile wheat 4.1; 8=mercantile wheat 4.2; 9= mercantile wheat 2.1; 12=mercantile
wheat 11.2; 14=cultivar Ljiljana 2; 15=mercantile wheat 6; 16=mercantile wheat 7; 18= artificially
contaminated wheat seed with Tilletia caries; 19=mercantile wheat 14
Distribution and diversity of Tilletia species in Vojvodina. Distribution and diversity of Tilletia
species in Vojvodina are shown in Fig. 4. Tilletia tritici was found in 127 samples in all examined
municipalities, whereas T. foetida was found in 11 samples in eight municipalities of Vojvodina.
Teliospores of Tilletia spp. which were assumed to belong to T. contraversa, T. bromi and T. fusca var.
fusca were found in 12 seed samples in seven municipalities of Vojvodina.
Figure 4 Diversity and distribution of Tilletia species in Vojvodina
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Current Status of Tilletia Species in Vojvodina, Serbia
Conclusions
Identification of Tilletia species based on morphological characteristics of teliospores is unreliable,
and that is very difficult to accomplish when very few teliospores are present in seed sample.
Župunski et al. (2012) focused attention on the problem that can occur if samples of wheat export
shipments contain Tilletia spp. teliospores which are morphologically similar to those of quarantined
T. contraversa. They also pointed out that more attention should be focused on confirmation of the
presence of T. contraversa in Vojvodina, Serbia.
In addition, Župunski et al. (2012) revealed that seed quality testing to determine contamination
level of economically important Tilletia species could be compromised as a consequence of inability
to identify individual teliospores. Although bunt contamination level was quantified down to less
than one spore per seed by using real-time PCR and multiplex real-time PCR techniques (McNeil et
al., 2004; Tan et al., 2009) it was not possible to make distinction between T. caries, T. contraversa and
T. bromi. McDonald et al. (1999) were also unsuccessful in identification of individual teliospores of
Tilletia species by using primers designed for conserved binding sites in internal transcribed spacer
(ITS) region. Župunski et al. (2012) indicated that all molecular techniques which were successful in
distinguishing Tilletia species were established and confirmed by using Tilletia species collections or
large number of teliospores (Gao et al., 2010; Gao et al., 2011; Kellerer et al., 2006; Liu et al., 2009;
McDonald et al., 2000; Yuan et al., 2009). Therefore, there is a need for validation of these methods on
low contaminated wheat seed samples in order to make plant protection more efficient and reliable.
Acknowledgements
We are grateful to Dr. Mark Gleason (Department of Plant Pathology and Microbiology, Iowa State
University, USA) for useful suggestions and critical reading of the manuscript.
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