Characterization of Bipolaris sorokiniana and Alternaria sesami isolates obtained from sesame (Sesamum indicum L.) in Egypt

INTRODUCTION

 Sesame (Sesamum indicum L.) is one of the ancient crops and considered as most important oilseed crop in tropical and subtropical areas of the world. The cultivated sesame derives from wild populations originated from the South Asia, especially the western Indian peninsula, or the Punjab and parts of Pakistan (Fuller, 2003). Sesame is attacked by several infectious plant pathogens which are responsible for major damaging factor to crop plants. Among important sesame diseases, Alternaria leaf spot is prevalent in all the sesame growing areas of the world (Verma, et al., 2005; Kolte, 1985; Bhale, et al., 1998). The Alternaria leaf spot, caused by Alternaria sesami (Kawamura) Mohanty and Behera, produces small, irregular brown spots on the leaf blade. These spots collapse each other and form elongated lesions. Same symptoms may also appear on stem and seed capsule. Due to severe infection, the plants may become defoliated completely. Species of Bipolaris (Shoemaker, 1959), are important plant pathogens associated with over 60 host genera (Sivanesan, 1987; Manamgoda, et al., 2011; Agrios, 2005).

Accurate identification and precise naming of species are crucial as the name is the key to accessing all accumulated knowledge (Cai, et al., 2009 and 2011; Hyde, et al., 2010; Hawksworth, et al., 2011; KoKo, et al., 2011; Udayanga, et al., 2011). Using molecular taxonomy, many uncertainties of conventional taxonomy can be resolved (Valente, et al., 1999; Mendoza, et al., 2001). Phylogenetic studies of Bipolaris have shown that the genus Bipolaris is not monophyletic and some Bipolaris species with short straight conidia cluster with Curvularia (Goh, 1998; Berbee, et al., 1999; Emami and Hack, 2002; Manamgoda, et al., 2011).

DNA markers have become a powerful tool to study taxonomy and molecular genetic of a variety of organisms. The random amplified polymorphic DNA (RAPD) allows quick assessment of genetic variability in various taxa, and has been used to study inter and intraspecific variability among isolates of several fungal species (Inglis, et al., 2001 and Tigano-Milani, et al., 1995a, b).

Clear understanding of the extent of variation in virulence would be helpful in developing plant cultivars with stable resistance. In this regard, several studies were carried out to analyze the aggressiveness of Bipolaris sorokiniana and Alternaria sesami among isolates collected from different agroecological zones. Controlling plant disease with the use of cultural practices and resistant varieties are considered as safest control measures. In this regard many research workers from elsewhere evaluated sesame germplasm against the Alternaria sesami for the search of resistant cultivars (Jayaramaiah et al., 1981; Shekharappa and Patil, 2001; Basavaraj et al., 2007).

Keeping in view the losses caused by the leaf spot disease of sesame, the present study was carried out to characterize fungal pathogenic causes leaf spot disease of sesame in Egypt. Also, the aggressiveness, varietal response and host range of the both causal fungi were studied.

MATERIALS AND METHODS

Sampling and pathogens isolation

Sample collection.

Diseased sample of sesame plant were collected from sesame plantations located in four localities that belonged to three governorates during the summer of 2016. The sampling localities were: Abou-Hommos, Hosh Isa (Al-Buhayrah Governorate), Tamia (Al-Fayoum governorate) and Beni Suef (Beni Suef Governorate). Symptomatic plants were carefully investigated and the regarded samples (leaves, stem and capsules) were collected and potted in new clean plastic pages then transported to the laboratory.

Isolation of the causal pathogens

In isolation processes, the samples were washed under running tap water for 2 minutes and then excised into small pieces (0.5 cm). These pieces were disinfested in 0.1% Sodium hypochloride for 2 min then rinsed in sterilized distilled water and dried between two sterilized filter papers. The surface sterilized samples were plated onto Potato Dextrose Agar (PDA) medium and incubated at 27°C until the fungal growth was appeared. The resulted fungal growths were transferred to new PDA pleats and then purified using hyphal tip or single spore techniques then labeled and kept on PDA slants at 5°C for further studies.

 Identification of isolated fungi:

Cultural and microscopic characteristics.

Cultural and microscopic characters of the obtained isolates were studied according to (Alcorn, 1983 and 1988; Sivanesan, 1987). The colony morphology (texture, colour and growth type) and microscopical features (production of conidia, conidial and conidiophore morphology and hyphal septation) were checked and recorded. Spore morphology was studied by examining the conidial spores obtained from mature pure culture of the regarded fungal isolate under low and high-power objective (10x and 40x) of microscope. Measurements of the fungal dimension in terms of length, breadth, beak length and number of septa were determined using stage and ocular micrometer. The average of length and breadth of the conidial body, beak and number and number of transverse and longitudinal septa were recorded from 25 mature conidia for each isolate. These observations were compared with those of the standard measurements given by (Simmons, 2004). The microphotographs were taken to show the spore morphology.

Pathogenicity test

The pathogenicity of the selected fungal isolates was demonstrated by artificially inoculation of sesame plants by spore suspension along with mycelial fragments obtained from 10 days old cultures of these isolates. Pure cultures of the selected isolates were grown on potato dextrose agar medium (PDA) for 10 days. The fungal suspension of each isolate was prepared by placing 20 mL of sterilized distilled water in the culture plate and swabbed using paint brush and the resulted suspension were completed to 100 ml. The suspension (2×10³) of each isolate were sprayed using manual atomizer on the foliage of 1month old healthy sesame plants which were grown on plastic pots that were potted with sterilized mix of Peat Moss and sand. In the same manner, plants of same age were sprayed with sterile water only and served as control. The treated plants were kept for 2 days in a humid plastic bag. The tested plants were observed daily and any external symptoms was recorded.

Molecular studies

For molecular identification one isolate from each of A. sesami and B. sorokiniana were chosen to confirm their identity using Internal transcribed spacer (ITS) technique (White, et al., 1990), the investigation was carried out in the Genetics Microbial Laboratory at the Agriculture Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, Egypt.  The details of the test as follows:

DNA extraction and purification:

DNA extraction and purification were carried out according to DNeasy Kit (Qiagen). The fungal mass was grinding under liquid nitrogen to a fine powder using a mortar and pestle. The powder and liquid nitrogen were transferred to sterilized test tube and left until the liquid nitrogen was evaporated. After this step, a 400 µL of buffer AP1 and 4 µL of RNase A stock solution (10 mg/mL) was added to 100 mg of ground fungal mass and vigorously vortexed. To lyses the cells, the mixture was incubated for 30 min at 65°C with inverting the tub 3 times during incubation to mix the mixture. A 130 µL of buffer AP2 was added to the lysate mix and incubate for 5 min on ice to precipitate detergent, proteins, and polysaccharides. The lysate was applied to the QIAshredder spin column (lilac) sitting in a 2 mL collection tube and centrifuged for 2 min at maximum speed. The component was transferred to a new tube without disturbing the cell-debris pellet. A 1.5 volumes of buffer AP3/E were added to the cleared lysate and mixed by pipetting. A 650 µL of this mixture, including any precipitate which may have formed, was applied to the DNeasy mini spin column sitting in a 2 mL collection tube (supplied) and centrifuged for 1 min. at 8000 rpm and discard flow-through. The DNeasy column was placed in a new 2 mL collection tube (supplied) and 500 µL of buffer AW was added to the DNeasy column and centrifuged for 1 min. at 8000 rpm and then discard flow-through and the collection tube was reused in the next step. A 500 µL buffer AW was added to the DNeasy column and centrifuged for 5 min. at maximum speed to dry the membrane. The DNeasy column was transferred to a 1.5 mL microcentrifuge tube (not supplied) and 100 µL of preheated (65°C) buffer AE was pipetted directly onto the DNeasy membrane. The tube was incubated for 5 min. at room temperature and then centrifuged for 1 min at 8000 rpm to elute. Elution with 50 µL (instead of 100 µL) increases the final DNA concentration in the eluate significantly.

Estimation for the DNA concentration:

A 2 µL of the parent’s DNA samples was run on 1% agarose gel in comparison to 10 µL of a DNA size marker (lambda DNA Hind III digest Phi X 174/ HaeIII digest). To estimate DNA concentration, the degree of fluorescence of the DNA sample was compared with the different bands in DNA size marker.

Polymerase Chain Reaction (PCR) using ITS:

The first round of amplification was performed using universal eukaryote primers ITS1 (F 5′- TCCGTAGGTGAACCTGCGG -3′) and ITS4 (R 5′TCCTCCGCTTATTG ATATGC-3′) according to (White, et al., 1990).

PCR components:

The PCR amplification was performed in a total volume of 50ul, containing 1X reaction buffer (10 m M Tris-HCl, pH 8.3 and 50 m MKCl), 1.5 m M MgCl₂, 1U Taq DNA polymerase (promega), 2.5m M dNTPs, 30 poml of each primer and 30 ng genomic DNA.            

PCR condition (thermal profile):

PCR amplification was carried out in a Perkin-Elmer/ Gene Amp® PCR System 9700 (PE Applied Biosystems) thermocycler. The PCR amplification conditions were as follows: an initial extended step of denaturation at 94°C for 5 min followed by 40 cycles of denaturation at 94°C for 30 Sec., primer annealing at 50°C for 30 Sec. and elongation at 72°C for 1 min. The primer extension segment was extended to 7 min at 72°C in the final cycle.

Purification of PCR Products:

Amplified products from fungal isolates were purified using EZ-10 spin column PCR products purification PCR reaction mixture was transferred to 1.5 mL microfuge tube and three volumes was added of binding buffer 1 after that the mixture solution was transferred to the EZ-10 column and loft room temperature for 2 min. after that centrifuge, 750 µL of wash solution was added to the column and centrifuge at 10.000rpm for two minutes, repeated washing, 10.000 rpm was spine for an additional min. to remove any residual wash solution. the column was transferred into a clean 1.5 mL microfuge tube and add 50 µL of elution buffer, incubated at room temperature for 2 minutes and when store purified DNA at -20 °C.

ITS-sequencing analysis:

A representative fungal isolate was selected for sequencing analysis. A part of the rDNA region ITS1–5.8S rDNA–ITS4 was amplified using the forward (ITS1) primer pairs. The sequencing of the product PCR was carried out in an automatic sequencer ABI PRISM 3730XL analyzer using Big Dye TM Terminator Cycle Sequencing Kits following the protocols supplied by the manufacturer. Single-pass sequencing was performed on each template using ITS1 primer. The fluorescent-labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The samples were resuspended in distilled water and subjected to electrophoresis in an ABI 3730xl sequencer (Macrogen).

Computational analysis (BLASTn) ITS.

The sequences were analyzed using BLAST program (http://www.ncbi.nlm.nih. Gov/ BLAST) Sequences were aligned using Align Sequences Nucleotide BLAST.

Comparison between isolates using RAPD-PCR Reactions:

A set of 14 primers RAPD (Table, 1) was used to detection the polymorphism among the 5 fungal isolates of Alternaria (AM1, AM2, AM5, AM6 and AM11) and the 5 fungal isolates of Bipolaris (BM3, BM8, BM19, BM20 and BM22). The amplification reaction was carried out in 25 μL reaction volume containing 1X PCR buffer, 1.5 mM MgCl2, 0.2 m MdNTPs, 1 μM primer, 1 U Taq DNA polymerase and 30 ng template DNA.

Thermocycling Profile and Detection of the PCR Products:

PCR amplification was performed in a Perkin – Elmer/ Gene Amp® PCR System 9700 (PE Applied Biosystems) programmed to fulfill 35 cycles after an initial denaturation cycle for 5 min at 94°C. Each cycle consisted of a denaturation step at 94°C for 1 min, an annealing step at 36°C for 1 min, and an elongation step at 72°C for 1.5 min. The primer extension segment was extended to 7 min at 72°C in the final cycle. The amplification products were resolved by electrophoresis in a 1.5% agarose gel containing ethidium bromide (0.5µg/mL) in 1X TBE buffer at 95 volts. A 100bp DNA ladder was used as a molecular size standard. PCR products were visualized on UV light and photographed using a Gel Documentation System (BIO-RAD 2000).

Table 1. Sequence of the fourteen arbitrary primers RAPD - PCR.

Data analysis

The banding patterns generated by RAPD -PCR marker analyses were compared to determine the genetic relatedness of the samples under study. Clear and distinct amplification products were scored as ‘1’ for presence and ‘0’ for absence of bands. Bands of the same mobility were scored as identical. The genetic similarity coefficient (GS) between two genotypes was estimated according to Dice coefficient (Sneath and Sokal, 1973).

Dice formula: GSij = 2a / (2a+b+c )

Where GSij is the measure of genetic similarity between individuals i and j, a is the number of bands shared by i and j, b is the number of bands present in i and absent in j, and c is the number of bands present in j and absent in i. The similarity matrix was used in the cluster analysis. The cluster analysis was employed to organize the observed data into meaningful structures to develop taxonomies. At the first step, when each accession represents its own cluster, the distances between these accessions are defined by the chosen distance measure (Dice coefficient). However, once several accessions have been linked together, the distance between two clusters is calculated as the average distance between all pairs of accessions in the two different clusters. This method is called Unweighted Pair Group Method using Arithmetic Average (UPGMA) (Sneath and Sokal, 1973).

 Host rang

Pathogenic capability of Bipolaris sorokiniana and Alternaria sesami isolates were tested on 11 plant species belonged to three families. The tested plants were: Sorghum, Maize, Wheat, Barley, Rice, Tomato, Broad bean, Datora, Potato, Pepper and Bean. The tested plants were sprayed to runoff with 100 mL of 106 conidia /mL of conidial suspension of appropriate isolate and loosely enclosed in a plastic bag for 24 h. The inoculated plants were kept under open field conditions. Ten plants from each plant species were used for each isolate. The reactions of the inoculated plants were monitored daily for 4 weeks after inoculation date.

Isolates aggressiveness.

Aggressiveness of five identified isolates was evaluated on leaves of sesame plants (variety Shandawil-3) after artificial inoculation using spraying method. Inoculum of each fungal isolates were prepared as mentioned above. The tested plants were sprayed to runoff with 100 mL of fungal suspension of appropriate isolate and loosely enclosed in a plastic bag for 24 h. The inoculated plants were kept in a greenhouse in which the average of temperature was 28°C. Ten plants were used for each isolate and the assays were repeated three times and the results of all tests were averaged and reported. The reactions were evaluated 20 days after inoculation according to the following indices:

 *Disease index (0-4) For Alternaria severity assessment.

0= No symptoms.

1= Tiny spots with number < 20 spots / leaf.

2= Small sized spots (< 0.2mm) with number >20 and < 60 spots /leaf.

3=Medium sized spots (>2mm) spots with total spot number > 60 and

4= As in three, but spots number > 120 spot / leaf, leaves defoliation and plant collapse.

*Disease index (0-4) For Bipolaris severity assessment.

0= No symptoms.

1= Small sized spots (< 0.5mm) with number < 20 spots / leaf.

2= Medium sized spots (>5mm) with number >20 and < 60 spots /leaf.

3= Extended blotches (>5mm) with total number > 60 / leaf.

4= As in three with severe defoliation and plant collapse.

Cultivar reaction

The pathological response of three sesame cultivars (viz., Shandawil-3, Suhaj-1 and Giza-32) to infection by Alternaria sesami and Bipolaris sorokiniana were tested in pots experiment. The conidial suspension was prepared as described in pathogenicity test. The number of conidia were counted using the hemocytometer and adjusted to 2×10³ conidia/mL (Ojiambo, et al., 1998). Forty days old plants of each variety were inoculated and kept as described earlier (in pathogenicity test). Twenty days after inoculation the disease severity was recorded as previously.

Statistical analysis

It's worth to be noted, the experiments of varietal reaction and isolates aggressiveness was designed as complete blotted design with five replicates for each variety. Data were subjected to ANOVA by using (Anonymous, 1988) and significant difference among the treatments was portioned by LSD test multiple range test at probability levels of p = 0.05.

RESULTS

Pathogens isolation

Isolation from symptomatic material of sesame plant on PDA medium yielded many fungal colonies growth with various cultural characteristics. After purification processes, the fungal isolates suspected to be causal agent of leaf spot were selected and non-regarded isolates were discarded. Generally, 19 fungal isolates were selected. The selected isolates were kept at 5 ^°C on PDA slant to accomplish the study.

Identification of the fungal pathogens

Cultural and microscopic characteristics:

Based on the characters of colony and morphological characters of conidiophores and conidia, the selected fungal isolates were restricted in the fungal genera of Alternaria and Bipolaris. The measurements of conidiophores and conidia were also found comparable with the standard measurements of Alternaria sesami and Bipolaris sorokiniana given in literatures.

On PDA medium, the Alternaria isolates colonies were fast growing, appeared initially as white with granular texture and then turning to greenish grey colour (Fig. 1). Aged culture appeared completely black with no aerial mycelium. In microscopically observation, the conidiophores of the fungal isolates were simple bearing acropetal chains (blastocatenate) of multicellular conidia at the apex. Conidia were pale brown colored, obclavate, with a hyaline beak (Fig. 2). The number of horizontal and vertical septa varied from four to12 and zero to six respectively.

In the same medium, the Bipolaris isolates varies in morphology, from dark olive-brown to grayish suppressed mycelial growth (Fig. 1). In microscopically observation, simple conidiophores are produced in fresh culture, and may be single or clustered. Conidiophores measure 6–10 x 110–220 µm with 6–11 septa. The conidia (Fig. 2), are olive-brown and elliptical with 5 to 9 cells and measure 15–28 x 40–120 µm. They arise from pores beneath each conidiophore septum and exhibit bipolar germination, or development of hyphae from each end of the spore (Fig. 3).

Pathogenicity test

Molecular identification (ITS rDNA gene)

PCR using primer pairs ITS1/ITS4 yielded specific species band of amplification product for both fungal isolates (Fig. 5). ITS pattern of Fungal isolates was subjected for ITS sequence analysis. BLAST analysis of the nucleotide sequence of the ITS region of Alternaria isolate showed 99% similarity with Alternaria sesami strains. whereas BLAST analysis of the nucleotide sequence of the ITS region of Bipolaris isolate showed 93% similarity with Bipolaris sorokiniana.

Randomly Amplified Polymorphic DNA (RAPD)

Polymorphism detected by RAPD markers

Fourteen RAPD primers were screened with the DNA of the fungal isolates to detect the Polymorphism among these isolates. Five isolates of Alternaria (AM1, AM2, AM3, AM4 and AM5) and five isolates of Bipolaris (BM1, BM2, BM3, BM4 and BM5) were screened separately. These produced multiple band profiles with a number of amplified DNA fragments.

In the case of Bipolaris isolates, multiple band profiles with a number of amplified DNA fragments ranging from 8 to 19 were produced (Table, 2 and Fig. 6). The total number of fragments produced by the 14 primers was 168 with an average of 12 fragments/primer. While, the number of polymorphic fragments ranged from 2 to 14. A maximum number of 19 amplicons were amplified with primer OPA-20, while the minimum number of fragments (8) was amplified with primers OPB-17. The highest number of polymorphic bands (14) was obtained with primers OPA-06, which exhibited the highest percentage (100%) of polymorphism. also revealed that the total number of polymorphic amplicons obtained by the fourteen studied primers was 100. This corresponds to a level of polymorphism of 57% and an average number of polymorphic fragments/primers of 7.1.

In the case of Alternaria isolates, multiple band profiles with a number of amplified DNA fragments ranging from 8 to 16 were produced (Table, 3 and Fig. 7). The total number of fragments produced by the 14 primers was 147 with an average of 10.5 fragments/primer. While, the number of polymorphic fragments ranged from 1 to 9. A maximum number of 16 amplicons were amplified with primer OPA-20, while the minimum number of fragments (8) was amplified with primers (OPA-05, OPA-08, PB-12 and OPB-17). The highest number of polymorphic bands (9) was obtained with primers OPA-20, which exhibited the highest percentage (56%) of polymorphism. Also revealed that the total number of polymorphic amplicons obtained by the 14 studied primers was 52. This corresponds to a level of polymorphism of 36% and an average number of polymorphic fragments/primers of 3.7.

Table 2. Primer name, Primer sequence, Total bands (TB), monomorphic bands (MB), polymorphic bands (PB), percentage of polymorphism (%P), Frequency (F) and size detected by RAPD.

Table 3. Primer name, Primer sequence, Total bands (TB), monomorphic bands (MB), polymorphic bands (PB), percentage of polymorphism (%P), Frequency (F) and size detected by RAPD Alternaria sesami.

Genetic relationships among the five fungal isolates as revealed by RAPD Markers:

To examine the genetic relationships among each group of fungal isolates based on RAPD results, the scored data were analyzed using the dice coefficient to compute the similarity matrices. These similarity matrices were used to generate a dendrogram using the UPGMA method. In the case of Bipolaris isolates, the estimated genetic similarities ranged from 74% to 82% revealing very high levels of genetic similarity among the studied accessions. The highest genetic similarity percentage (82%) accrued between isolate 8 and isolate 20, while the lowest genetic similarity (74%) was detected between isolate 3 and isolate 8. In the case of Alternaria isolates, the estimated genetic similarities ranged from 85% to 96%. This also revealing very high levels of genetic similarity among the studied accessions. The highest genetic similarity (96%) accrued isolate 2 and isolate 5, while the lowest genetic similarity (85%) was detected between isolate 1 and isolate 5 and between isolate 1 and isolate 6.

Host range:

Pathogenic capability of Bipolaris sorokiniana and Alternaria sesami isolates were tested on 11 plant species belonged to three families (Table, 4). In general, the isolates varied in its pathogenicity on the tested plants. The results showed that, all isolates of Bipolaris sorokiniana were pathogenic on tomato, maize, broad bean, bean, sorghum and wheat. Also, none of these isolates produced any symptoms on the tested plants. On the other hand, the isolates of Alternaria sesami markedly varied in its pathogenicity on the tested plants. Prove this, isolates AM1 and AM11 produced symptoms on one plant (tomato in the case of AM1 and bean in the case of AM11); isolate AM2 produced symptoms on two plants (broad bean and sorghum); isolates AM5 and AM6 produced symptoms on four plants (tomato, broad bean, bean and sorghum).

Table 4. Host range of B. sorokiniana and Alternaria sesami on different plants species.

+= Pathogenic; - = Nonpathogenic.