Analysis of virulence and genetic variability of Alternaria alternata associated with leaf spot disease in potato plants in Iran

Leaf spot disease in potato is caused by Alternaria alternata (Fr.) Keissler, an opportunistic pathogen that infests many agricultural crops worldwide in the field and during postharvest storage of vegetables and fruits. Alternaria alternata is associated with leaf spot disease in potato in Iran. Thus, there is a need to investigate the virulence and genetic variability of Iranian A. alternata isolates to facilitate the development of appropriate management strategies. In the present study, we analyzed a total of 28 isolates obtained from the main potato-growing regions of Iran, including the Ardebil, Hamedan, Isfahan, and Fars provinces. The pathogens were characterized based on sequence analysis of the genes encoding glyceraldehyde3-phosphate dehydrogenase (gpd), plasma membrane ATPase, Alternaria allergen a 1 (Alt a1), calmodulin, and actin. In addition, random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), and virulence studies were performed. Phylogenetic analysis of the combined dataset indicated that the five representative isolates were grouped with the subcluster comprising A. alternata. RAPD and ISSR analyses clustered the 28 A. alternata isolates into different groups with no correlation with their corresponding geographical origins. Results of the pathogenicity assay indicated that all A. alternata isolates were pathogenic against potato. However, the A. alternata isolates showed high variability in terms of virulence.


Introduction
Alternaria alternata (Fr.)Keissler has been isolated from a wide range of crops, including fruits and vegetables, nuts, and cereals [1]. A. alternata is a common saprobe found in many plants and other substrata worldwide and is an opportunistic pathogen that infects many agricultural crops in the field and during postharvest storage of vegetables and fruits.In potato, the A. alternata fungus causes brown necrotic lesions in foliage and black pit [2].Previous reports have shown that A. alternata can destroy more than 20% of potato production [3,4].In Iran, two species, namely, A. alternata and A. solani (Ellis & Martin), have been identified as the causal agents of leaf spot disease in potato, with A. alternata being the dominant species [5].
In recent years, multigene phylogeny has been widely employed for identification and characterization of Alternaria species [6,7].Molecular approaches based on sequence analysis of gene fragments encoding glyceraldehyde-3-phosphate dehydrogenase (gpd), plasma membrane ATPase, Alternaria allergen a 1 (Alt a1), calmodulin, and actin have robustly defined the monophyly of Alternaria in the ascomycete family Pleosporaceae [5].The efficacy of control strategies on the plant pathogen populations have been hampered by limited information on genetic variability [6,8,9].The most commonly adopted control measure is the use of fungicides and resistant cultivars.In Iran, genetic information is available based on ISSR of only 11 A. alternata isolates from potato plants collected in Hamedan Province.Moreover, no comprehensive study has analyzed the genetic diversity among the Iranian A. alternata isolates from various potato-growing regions for control planning programs [10].Therefore, given the decline of resistance of potato plants to A. alternata and the lack of appropriate breeding programs, there is an urgent need to study the genetic variability within pathogen populations and should be considered one of the first steps for disease management strategy of new and aggressive isolates and evaluation of the resistance of different potato varieties [11].Thus, considering the importance of leaf spot disease in potato and A. alternata in Iran [12], efforts should focus in investigating the present status of the disease.
Therefore, the present study aimed to estimate the variability among A. alternata isolates obtained from potato plants collected from the main potato growing regions of Iran.We aimed to obtain updated and more reliable information on the virulence and genetic variability of A. alternata populations, which could aid in the selection of appropriate breeding and management strategies against the A. alternata pathogen.

Fungal isolates
A total of 28 single-spore isolates [13] of A. alternata were obtained from the Plant Pathology Laboratory, Isfahan Research and Education Center for Agriculture and Natural Resources, Isfahan, Iran (Tab.1).The isolates were collected in 2010 from the main potato growing regions of Iran, namely, Ardebil, Hamedan, Isfahan, and Fars provinces (Fig. 1).Population samples of A. alternata were collected in June to August 2010 from randomly selected fields of potato plants from 28 sites in four provinces.For each sampling site, leaves with A. alternata lesions were collected from the potato plants.The A. alternata populations originated from seven isolates from every province, namely, the Ardebil, Hamedan, Isfahan, and Fars provinces.Conidia of all isolates were produced in chains on conidiophores and presented inverted pear shapes with short beaks and dark brown color, with dimensions of 20-50 µm × 9-18 µm and two to six (four) transversal and one to four (two) longitudinal septa.Each of the 28 isolates were confirmed to be A. alternata based on the morphological characteristics [4].

Virulence assay
Pathogenicity tests were conducted in a greenhouse using all 28 A. alternata isolates.The experiment was carried out based on a completely randomized design with 10 replicates (pots) for infected plants and control plants following the methods described by Mamgain et al. [8].Potato (Solanum tuberosum) ' Agria' seed tubers were planted in plastic pots (30 cm diameter) containing soil and perlite under greenhouse conditions (18-25°C) at the Plant Protection Research Department, Isfahan Agricultural and Natural Resources Research and Education Center, (AREEO), Isfahan, Iran.Potato tuber seeds consisted of small whole tubers weighing approximately 50 g each.One tuber seed piece was planted in each pot.Tubers were treated with thiabendazole (tekto WP60%, Golsam Gorgan Company, Tehran) before planting.Plants were irrigated once per day or as needed.Each replication consisted of ten leaves of each plant.Conidial suspension spray (103 spores/mL) was used to inoculate 1-month-old potato ' Agria' plants after emergence.After pathogen inoculation, the plants were covered for 48 h with plastic bags in the greenhouse with 14 h of light and 10 h of darkness [14].For estimation of the proportion of the leaf surfaces infected with A. alternata, disease rating was conducted at 7 days after inoculation based on the following 5-point scoring scale: 0 = no disease symptoms, lesions as pinpoints and nonmeasurable; 1 = <10% of the leaves have brown necrotic lesions; 2 = 10% to 25%; 3 = more than 25% to 50%; 4 = more than 50% to 75% of the leaves have brown necrotic lesions; and 5 = more than 75% to 100% of the leaves have brown necrotic lesions or appeared completely brown [15].The experiments were repeated twice, and reisolation of the inoculated fungi was performed to fulfill Koch's postulate.The following formula was used to calculate percent disease severity (PDS) in each replication: PDS = (∑RT × 100)/(SN), where T represents the total number of leaves in each category, R is the disease severity scale, N is the total number of leaves tested, and S is the highest number in the scale.
Data were transformed to arcsine square root and analyzed by conducting analysis of variance procedure (ANOVA, p < 0.05).Means were compared by performing Duncan's multiple range test using SAS software version 9.2.

DNA extraction, PCR amplification
Total genomic DNA was extracted from all isolates using the CTAB method [16].A NanoDrop ND-1000 spectrophotometer (LMS Co., Ltd, Tokio, Japan) was used to verify the quality and concentration of genomic DNA extracts.To characterize the isolates, the following genes were PCR-amplified using the indicated primers: a region of the gpd gene using the primers gpd1 and gpd2 [17], Alt a1 using primers Alt-a1-for and Alt-a1-rev [18], calmodulin using primers CALDF1 and CALDR1 [5], actin using primers ACTDF1 and ACTDR1 [5], and plasma membrane ATPase using primers ATPDF1 and ATPDR1 [5].PCR amplification of the gene sequences was performed as described by Lawrence et al. [5].PCR products were then purified using the Gene JETTM commercial PCR Purification Kit (Fermentas, Axon Scientific, Malaysia) and sequenced using the same forward and reverse primers by a commercial sequencing service provider (Iraizol Old Extraction DNA Kits, Rona Bio-Fanavaran Co., Iran).

Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses
Based on the results of initial screening against a set of representative isolates (Tab.2), we selected six out of 15 primers, namely, OPA-  Tab. 2 RAPD and ISSR primers utilized to identify and assess interspecific genetic diversity among Alternaria alternata isolates collected from potato plants.

RAPD and ISSR analyses
All PCR runs were performed in triplicate to confirm the consistency of amplification.The bands were considered as binary characters and were scored as either 1 (presence of DNA band) or 0 (absence of DNA band).The scores were then entered into a matrix and analyzed based on the numerical taxonomy and multivariate analysis using the program NTSYS-pc 1.8 (Applied Biostatistics Inc., Setauket, NY, USA) [19].
The similarity matrix was calculated using Jaccard's similarity coefficient.Clustering was performed using the unweighted pair group method using arithmetic averages (UPGMA) to generate the dendrogram.

Sequence alignment and phylogenetic analysis
For molecular identification, five representative isolates, namely, Aa-A5, Aa-A6, Aa-H2, Aa-H3, and Aa-I1, were selected from the 28 isolates.One isolate was selected from each of the five groups based on the RAPD and ISSR grouping.Consensus sequences were generated from the forward and reverse DNA strands using BioEdit Sequence Alignment Editor [20].To analyze the phylogenetic relationships between the A. alternata isolates and other Alternaria species, the sequences of the five representative isolates from this study and sequences of all the reference of the species-groups of Alternaria species [5] were aligned using the Clustal W multiple alignment program [21].Stemphylium callistephi was used as the out-group taxon.We performed phylogenetic analysis of the combined dataset comprising the gpd, plasma membrane ATPase, Alt a1, calmodulin, and actin genes using maximum likelihood method with the Kimura two-parameter model in MEGA 7.0 (GenBank accession, Fig. 2) [22].Branch support of the trees obtained from maximum likelihood analysis was assessed by bootstrapping with 1,000 replications to estimate the reliability of inferred monophyletic groups.MEGA7: Molecular Evolutionary Genetics Analysis version as used 7.0 for larger datasets [22].

Virulence assay
Symptoms of the leaf spot disease were observed in all the inoculated potato plants at 7 days after inoculation.Foliar lesions were initially visible as pinpoints that were irregular to circular in shape.Brown spots were usually observed first on the lower leaves and subsequently spread as circular spots throughout the leaves.Control plants did not exhibit any symptoms of the disease.Alternaria alternata isolates were reisolated from inoculated plants and were found to be identical to the original isolates used.These results reconfirmed the A. alternata isolates as the causal agents of leaf spot disease in the potato plants.However, we observed high variability in virulence among the four isolates (Tab.1).The Aa-F4 (PDS of 69.17) isolate was found to be the most virulent isolate, while the three isolates, namely, Aa-H6 (PDS of 12.50), Aa-A1 (PDS of 16.50), and Aa-A4 (PDS of 17.67), were the least virulent isolates.Other isolates showed moderate virulence towards potato ' Agria' plants with no significant differences in virulence.

RAPD analysis
Amplification using six RAPD primers generated a total of 47 consistently amplified fragments (100 to 3,000 bp), of which 100% were polymorphic.The average number of bands per primer was 7.83.The dendrogram produced using UPGMA analysis based on Jaccard's coefficient indicated relatively high variability among the 28 A. alternata isolates.The Jaccard's similarity coefficient index ranged from 100% to 60%, with the mean value of 0.80 (Fig. 3).The genetic diversity showed no association with the geographical origin of the isolates.

ISSR analysis
The nine ISSR primers generated a total of 61 consistently amplified DNA bands, of which 83.61% were polymorphic.The average number of bands per primer was 6.8.The bands ranged in size from approximately 100 to 3,000 bp.The dendrogram also indicated high variability among the 28 A. alternata isolates and grouped the isolates into different groups, with no correlation with the geographical regions of the isolates (Fig. 4).The Jaccard's similarity coefficient ranged from 100% to 68% with the mean value of 0.84.

Phylogenetic analysis
PCR amplification of the genes corresponding to gpd, plasma membrane ATPase, Alt a1, calmodulin, and actin in the five representative isolates produced fragments with sizes 797, 1,194, 467, 780, and 939 bp, respectively.Phylogenetic analysis of the combined dataset indicated that the five representative isolates were clustered in a subdistinct cluster, which included A. alternata with a high bootstrap value of 94% (Fig. 2).The five isolates were clearly distinguished from other selected Alternaria species, which were found to be highly similar to the A. alternata isolates in the present study.
Stemphylium callistephi was phylogenetically distant to Alternaria and clustered as its sister taxon.

Discussion
Results of the virulence assays indicated that all the A. alternata isolates were pathogenic against potato ' Agria' plants, and disease symptoms were observed in the leaves of all potato ' Agria' plants inoculated with A. alternata.Reisolation of A. alternata from all the inoculated samples confirmed Koch's postulates.Therefore, all A. alternata isolates were verified to be the causal agents of the leaf spot disease in the potato ' Agria' plants.Our findings also revealed high variability in virulence among the A. alternata isolates.
In addition, the grouping of the isolates based on virulence was not found to be correlated with the results of RAPD and ISSR analyses and the geographical origins of the isolates.Therefore, further detailed studies must be conducted using a larger number of A. alternata isolates.Our current findings were consistent with those of previous studies on A. alternata isolates obtained from citrus hybrids [23], potato plants [10,24], and tomato plants [24,25] throughout Iran.The RAPD and ISSR techniques are extremely powerful tools for distinguishing among individuals that exhibit intraspecific and interspecific variability [26].In the present study, 100% and 84% of the isolates were found to be polymorphic with respect to the RAPD and ISSR markers, respectively.The similarity indices were 84% and 80% for the RAPD and ISSR markers, respectively.RAPD and ISSR markers exhibit lower polymorphism rates than those of AFLP and other molecular markers.However, the AFLP method is more laborious, time-consuming and costly and involves the use of radioactive reagents.Therefore, the RAPD and ISSR techniques could serve as reliable alternatives for molecular characterization because they are easier to implement and are less costly [9,24].The RAPD and ISSR methods have been employed to generate comprehensive information regarding the genetic variability of A. alternata populations in citrus [23], linseed [27], pistachio [28], potato [10,24], tangerine [13], and tomato [24,25].These methods have also been used to investigate the genetic variability of other fungal species, such as Stemphylium lycopersici, which is associated with leaf spot disease in vegetable crops [26].In the present study, the results of RAPD and ISSR analyses were very similar and showed that the markers were 84% to 100% polymorphic, respectively.Therefore, the 28 A. alternata isolates exhibited relatively high diversity in the main potato-growing regions of Iran.The RAPD and ISSR markers showed no correlation with the geographical origins of the isolates, suggesting that the isolates are widely dispersed across Iran.The above results were consistent with those of a previous study, in which ISSR markers were used to assess the genetic diversity of 11 A. alternata isolates that caused brown leaf spot disease in potato plants throughout Iran [10].Furthermore, our findings indicated that both RAPD and ISSR markers can be used to reliably evaluate the diversity of A. alternata isolates.
Recently, sequencing of different DNA regions has been used as an alternative to morphology-based identification of Alternaria species [5,7,29] and other fungal species.Consequently, different DNA regions have been used for the characterization and identification of various Alternaria species [5].Studies have indicated that the combination of various DNA genes, including gpd, plasma membrane ATPase, Alt a1, calmodulin, and actin, can successfully determine the monophyly of Alternaria species.The above-mentioned genes showed higher polymorphism than those of the translation elongation factor 1-alpha (TEF) and beta-tubulin genes.In the present study, phylogenetic analysis of the combined dataset comprising the gpd, plasma membrane ATPase, Alt a1, calmodulin, and actin genes confirmed that all five representative isolates, namely, Aa-A5, Aa-A6, Aa-H2, Aa-H3, and Aa-I1, were A. alternata species.Furthermore, we detected no significant differences in the sequences of the five target genes among the representative isolates.

Conclusions
In conclusion, RAPD and ISSR markers were successfully used to genetically distinguish all the A. alternata isolates.The results of the present study revealed the extant diversity in pathogenicity and genetics of A. alternata isolates.Therefore, new and aggressive isolates should be screened to evaluate the potential resistance of potato varieties, which would be useful for the selection of appropriate breeding strategies against the potato pathogen A. alternata.To our knowledge, no comprehensive study has investigated the genetic diversity of A. alternata isolates that cause leaf spot disease in potato plants from various geographical regions in Iran.RAPD and ISSR markers could also be used to investigate a broader range of A. alternata isolates from other regions of the world and can also be extended to other host plants.Given that temperature, wind speed, potato cultivar, and other factors can affect the population structure of fungi,

Fig. 1
Fig. 1 Map of Iran showing the locations of potato plantations where the 28 Alternaria alternata isolates were collected.The names of the isolates are based on the names indicated in Tab. 1.

Fig. 2
Fig. 2 Phylogenetic tree constructed based on the combined dataset (five target genes) using five representative isolates from this study and other Alternaria species retrieved from GenBank.Stemphylium callistephi was used as the out-group taxon.The bar indicates the number of nucleotide substitutions per site.Numbers of bootstrap support values ≥50% based on 1,000 replicates.
Origins and virulence variability of Alternaria alternata isolates used in this study.
Tab. 11Alternaria alternata isolates causing leaf spot of potato.2Valuesfollowed by the same letter in the column did not differ significantly (0.05 level) in Duncan's multiple range test.