International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 4, Issue 6, Dec 2014, 53-60
TJPRC Pvt. Ltd.

ASSESSMENT OF GENETIC DIVERSITY IN DIFFERENT ISOLATES OF

TALAROMYCES FLAVUS BY RAPD MOLECULAR MARKER
LALEH NARAGHI1, ASGHAR HEYDARI2, SAEED REZAEE3 & MOHAMMAD RAZAVI4
1,3

Department of Plant Pathology, College of Agriculture and Natural Resources, Science and Research Branch,
Islamic Azad University, Tehran, Iran

2,4

Department of Plant Diseases Research, Iranian Research Institute of Plant Protection (IRIPP), Tehran, Iran

ABSTRACT
Antagonistic fungus, Talaromyces flavus, mediates growth inhibition on many soil borne pathogenic agents by
producing glucose oxidase, chitinase and pectinase enzymes. In this study, genetic diversity of different T. flavus isolates
related to different cultivation regions of cotton, potato, tomato and greenhouse cucumber was determined by Random
Amplification Polymorphic DNA (RAPD) markers. Results showed that majority of isolates in RAPD groups belonged to
different regions of one host plant. However, most isolates in RAPD sub- groups belonged to the cultivation regions of
each crop. In electrophoresis patterns of PCR- RAPD products by using different primers, three molecular phenotypes
were observed. In first type, there were two fragments with 750 bp and 250 bp in size. In second type, there were the
fragments bigger than 250 bp and smaller than 750 bp as well as above-mentioned two fragments. However, there were the
fragments bigger than 250 bp and smaller than 750 bp in third type.
In electrophoresis patterns related to isolates obtained from cultivation regions of cotton, tomato and potato,
all three molecular phenotypes were observed but only third type was observed in those related to isolates obtained from
cucumber greenhouses. Based on the observation from first and second types in T. flavus isolates obtained from cultivation
regions of cotton, tomato and potato, it is concluded that these phenotypes may be related to glucose oxidase enzyme
because this enzyme is activated by glucose and T. flavus isolates with high glucose oxidase activity are observed only in
the rhizosphere of the plants such as cotton, potato and tomato where their root exudates contain high levels of glucose.

KEYWORDS: Genetic Diversity, Random Amplified Polymorphic DNA, Talaromyces flavus

INTRODUCTION
The Talaromyces flavus species (with asexual stage of Penicillium dangeardii Pitt) is characterized as one of the
antagonist fungi against the important soil-borne pathogens like Verticillium albo-atrum, Verticillium dahliae, Rhizoctonia
solani, Sclerotinia sclerotiorum, and Sclerotinia rolfsii (Madi et al., 1997; Klosterman et al., 2009; Inglis and Kawchuk,
2002; and Tjamos et al., 2000). This fungus is thermophile and found mainly in the soil of regions with tropical climate
(Pitt and Hocking, 2009).
T. flavus species was reported for the first time in a cotton field located in Karkandeh research station from
Golestan province in Iran, in 2003 (Naraghi et al., 2003). The experimental and greenhouse studies, which were conducted
regarding the inhibitory impact of T. flavus isolates in Verticillium wilt (V. albo-atrum and V. dahliae) in the above crops
indicated that the efficiency of these isolates has differed in terms of controlling the aforesaid agents (Naraghi et al., 2007;

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Laleh Naraghi, Asghar Heydari, Saeed Rezaee & Mohammad Razavi

Naraghi 2010; Naraghi et al., 2010a, b, and c).

No molecular study has been so far carried out on several T. flavus isolates in Iran including for determination of
genetic variation among them, but many investigations have been conducted abroad concerning to genetic variation on this
fungus as bio-controlling agent for Verticillium pathogenic isolates by means of RAPD method and for determining the
sequences of Internal Transcribed Spacer (ITS) regions of ribosomal DNA (Fravel and Bao, 2001; Chakrabotry et al.,
2011) as well as isolation of glucose oxidase gene from T. flavus and its role in biologic fighting against Verticillium wilt
disease (Murray et al., 1997).
The rate of antagonist activity of several isolates of T. flavus, which have been derived from major areas of
cultivating cotton, sugar beet, potato, tomato, and greenhouse cucumber in Iran, was determined against the important
soil-borne pathogenic agents including V. dahliae, V. solani, and Fusarium oxysporum among them, the most efficient
isolates have been also identified in terms of antagonist activity (Naraghi, 2010; Naraghi et al., 2010a, b and c; Naraghi et
al., 2010; Naraghi et al., 2010a and b). Thus, the present survey is intended to examine primarily the genetic variation of
several isolates of T. flavus so that following to these studies one could find specialized genes belong to isolates with the
maximum efficiency (yield) in terms of antagonist activity. Whereas application of indigenous isolates (Sahile et al., 2011)
is one of the important factors in successful biologic control in regions of cultivating of farming crops thus by expression
of such genes in certain farming plants one could strengthen the weak yield of their indigenous isolates (Murray et al.,
1999; Nguyen et al., 2011; Grover and Gowthman, 2003).

MATERIAL AND METHODS

T. flavus Isolates Preparation
At this phase, 60 isolates of T. flavus, which have been derived from major areas of cultivating cotton, potato,
tomato, and greenhouse cucumber and rate of their inhibitory activity with mycoparasitism, producing volatile and nonvolatile compounds for growing Verticillium wilt agent for the given crop (V. dahliae or V. albo-atrum) that was identified,
were used for this purpose. Among this quantity, 23 isolates belonged to cotton fields in Gorgan city (TF-Co-G-1
to TF-Co-G-11), Neishabur (TF-Co-N-12 to TF-Co-N-21), and Moghan (TF-Co-M-22 to TF-Co-M-23), and 15 isolates
were related to tomato fields in Varamin city (TF-To-V-24 to TF-To-V-33) and Oromiyeh (TF-To-U-34 to TF-To-U-38),
and 14 isolates belonged to tomato fields in Karaj (TF-Po-K-39 to TF-Po-K-47) and Varamin (TF-Po-V-48 to TF-Po-V52), and 8 isolates were related to cucumber greenhouse in Varamin (TF-Cu-V-53 to TF-Cu-V-60) (Naraghi, 2010) (Table
1).
Table 1: Talaromyces flavus Isolates Used in the Study

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Assessment of Genetic Diversity in Different Isolates of Talaromyces flavus by RAPD Molecular Marker

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DNA Extraction and Review on Genetic Variation of T. flavus Isolates by Random Amplified Polymorphic DNA
(RAPD)
DNA was extracted from T. flavus based on method of Mazzaglia et al. (Mazzaglia et al., 2001). In order to
examine genetic variation of the collected T. flavus isolates from cultivation regions of the studied farming crops
(cotton, tomato, potato, and greenhouse cucumber), 23 random primers were tested. Of the used primers, 13 primers
(UBC203, UBC208, UBC211, UBC213, UBC214, UBC215, UBC283, UBC285, UBC286, UBC772, UBC289, UBC726,
UBC736) were related to university of British Columbia (Vancouver, Canada) (Huang et al, 2005), 4 primers (OPR15,
OPE-07, OPA-03, OPA-02) belonged to (Alameda, California, Operon Technologies USA), and the remained primers
(31s, 29S, 17S, 16S, 12S, 4S) of S- series were related to Innsbruck Medical University (Innsbruck, Tyrol, Austria) (Yang
et al., 2011).
Optimization of RAPD-PCR
At this stage, DNA template primerof MgCl2 was optimized by means of dNTP-mix with various concentrations
in RAPD-PCR.
Polymerase Chain Reaction (PCR) Conditions
With respect to method of Williams et al. (Williams et al., 1990), PCR thermal cycle was conducted but with a
little change.
Electrophoresis of RAPD-PCR Product
The result of PCR product was treated by electrophoresis on agarose gel 1.2% consists of ethidium bromide
(0.5g/milt) in the electrophoresis device (model BIO-RAD GT, made in USA) and in tray with dimensions of (1624) at
90v for two hours. To determine size of fragments, marker (1kbm made in Germany) was utilized. The patterns derived
from Gel Documentation (Uvi Doc) radiographic device (Uvi Tec Inc, UK) and the given bands were evaluated. In order to
make sure of the acquired bands, PCR reaction was treated three times with each of the studied primers for DNA in each of
isolates of T. flavus.

RESULTS AND DISCUSSION

Extraction of DNA from T. flavus Isolates
DNA was extracted from 60 isolates of T. flavus. The calculated figure was acquired as 1.5-2 in optical absorption
at wavelength 280nm and this indicated the high purity of the extracted DNA. Concentration of DNA was regulated for all
isolates at approximately 25ng/l and used in the reactions.
Determination of Genetic Variation in T. flavus Isolates by Random Amplification of Polymorphic DNA (RAPD)
Technique
Among 23 random primers used in this study, 10 primers produced suitable reproducing products from DNA in
RAPD-PCR reaction and 196 fragments were derived totally where 91.32% of the generated fragments (179 fragments)
were polymorphic (Figures 1-4; Table 2). Based on the given results, three primers (UBC211, UBC213, and UBC286)
with 19- fragment polymorphisms (10.61% of total polymorphic fragments) had the maximum frequency of
polymorphism; while, primers UBC 203 and UBC283 with 16-fragment polymorphisms (8.93% of total polymorphic
fragments) indicated the minimum rate of polymorphism. With respect to comparison of the reproduced patterns in
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Laleh Naraghi, Asghar Heydari, Saeed Rezaee & Mohammad Razavi

electrophoresis gel, the isolates were classified in three groups. The first group had some fragments with sizes of 250 and
750 base pairs. Rather than two existing fragments in first group, four fragments were seen with sizes of 400, 500, 600, and
700 base pairs in the second group while the third group had only fragments with sizes of 400, 500, 600, and 700 base pairs
(Figures 1-4).
By observation of the given electrophoretic patterns, it can be concluded that primerUBC208 may appropriately
isolate isolates belong to potato from isolates of greenhouse cucumbers by reproduction of fragments with sizes of 3000,
4000, 5000, 6000, 8000, and 10000 base pairs (Figure 4). On the other hand, by employing this primerand presence of
such bands (strands) in the related isolates from Neishabur cotton fields (Figure 1), these isolates may be considered
genetically as similar to the derived isolates from potato fields. Similarly, primerUBC203 can be also isolated from the
tomato- related isolates by reproducing the given fragments in the isolates belonged to the cotton (Figure 2) and at the
same time with reproducing these fragments of the given isolates belonged to tomato fields in Oromiyeh, one could
appropriately separate them from the related isolates to tomato fields in Varamin region (Figure 3).
With respect to observation in band models of first and second groups in T. flavus isolates, which have been
derived from areas of cultivating cotton, tomato, and potato, it can be justified in this way that probably fragments with
sizes of 250bp and 750bp exist in first and second groups so this may serve as a marker to identify isolates with high
activity of glucose oxidase enzyme since this enzyme is only active with the presence of glucose and isolates with
highly- active glucose oxidase enzyme are found exclusively surrounding the root of plants in which secretions of their
root is full of glucose. According to the previous researches, sugar compounds like glucose and other polysaccharides exist
in rhizomatous secretions of cotton, tomato, and potato (Kumur et al., 2007; Krarchenko et al., 2003; Badri and Vivanko,
2009). Therefore, it is more likely that the isolates with highly-active glucose oxidase enzyme are further present
surrounding root of these plants while with respect to having antagonist effects in all the acquired isolates of T. flavus in
this investigation and the results of several studies regarding other produced enzymes by this fungus such as pectinase,
galactosidase, and lactonase (Crotti et al., 1999; Ayer and Racok, 1990), it is inferred that antagonist activity of the given
isolates from cucumber greenhouses has been affected by these types of enzymes.
The results of present study showed that the used primers are effective in recognition of T. flavus relating to a
certain host and or region. For example, primers UBC 203 and UBC208 were highly efficient respectively in identifying
the relevant isolates of T. flavus to cotton and potato hosts and primerUBC286 in recognition of the related isolates of T.
flavus to tomato fields in Oromiyeh. Similarly, some studies have been carried out on recognition of variety, sub-type, and
race in ascomycetes and basidiomycetes by means of ITS primers including ITS1, ITS1-F, ITS2, and ITS3 (Toju et al.,
2012). Likewise, Schmidt (Schmidt, 2009) indicated that Poly-T primermight recognize a certain form of Blumeria
graminis f. sp. hordei. Alternately, the findings of this investigation showed that there was genetic difference among
isolates of a host in various regions rather than genetic variation among the relevant T. flavus isolates to several hosts. In
this regard, Madi et al. (1997) acknowledged that there was difference in intensity of enzymatic activity and antagonist
efficiency (yield) among different isolates of T. flavus due to genetic variation among them.
With respect to band models, which were derived from isolates of T. flavus that belong to cotton, potato, and
tomato in this investigation at Oromiyeh by primers UBC203, UBC208, and UBC286, these primers can be used as
specialized primers for the above isolates in molecular studies in the future.

Impact Factor (JCC): 4.3594

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Assessment of Genetic Diversity in Different Isolates of Talaromyces flavus by RAPD Molecular Marker

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Figure 1: Electrophoresis Patterns of PCR- RAPD Products Using UBC208 Primer among Talaromyces flavus
Isolates, L: DNA Size Marker (1 Kb), Lane 2-11: T. flavus Isolates Related to Cotton Fields in Gorgan, Lane 12-15:
T. flavus Isolates Related to Cotton Fields in Neishaboor

Figure 2: Electrophoresis Patterns of PCR- RAPD Products using UBC203 Primer among Talaromyces flavus
Isolates, L: DNA Size Marker (1 Kb), Lane 16-21: T. flavus Isolates Related to Cotton Fields in Neishaboor, Lane
22-23: T. flavus Isolates Related to Cotton Fields in Moqan, Lane 24-29: T. flavus isolates Related to Tomato Fields
in Varamin

Figure 3: Electrophoresis Patterns of PCR- RAPD Products Using UBC286 Primer among Talaromyces flavus
Isolates, L: DNA Size Marker (1 Kb), Lane 31-33: T. flavus Isolates Related to Tomato Fields in Varamin, Lane 3438: T. flavus Isolates Related to Tomato Fields in Urumia, Lane 39-45: T. flavus Isolates Related to Potato Fields in
Karaj

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Laleh Naraghi, Asghar Heydari, Saeed Rezaee & Mohammad Razavi

Figure 4: Electrophoresis Patterns of PCR- RAPD Products Using UBC208 Primer among Talaromyces flavus
Isolates, L: DNA Size Marker (1 Kb), Lane 46-47: T. Flavus Isolates Related To Potato Fields in Karaj, Lane 48-52:
T. flavus Isolates Related to Potato Fields in Varamin, Lane 53-59: T. flavus Isolates Related to Cucumber
Greenhouses in Varamin
Table 2: The Number of the Polymorphic and Non-Polymorphic Fragments Produced by
10 Used Primers in PCR- RAPD Reaction

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