Distinction of Alternaria Sect. Pseudoalternaria Strains among Other Alternaria Fungi from Cereals

Species of the genus Alternaria are ubiquitous and frequently isolated from various plants, including crops. There are two phylogenetically and morphologically close Alternaria sections: the relatively well-known Infectoriae and the rarely mentioned Pseudoalternaria. Currently, the latter includes at least seven species that are less studied and sometimes misidentified. To perform precise identification, two primers (APsF and APsR) were designed and a sect. Pseudoalternaria-specific PCR method was developed. Thirty-five Russian A. infectoria-like strains were then examined. Five strains were found to be the members of the sect. Pseudoalternaria. Additionally, specificity of the previously developed primer set (Ain3F and Ain4R) was checked. It was found to be highly specific for sect. Infectoriae and did not amplify sect. Pseudoalternaria DNA. Identification of strains of the sect. Pseudoalternaria was supported and refined by phylogenetic reconstruction based on analysis of two loci, the glyceraldehyde-3-phosphate dehydrogenase gene (gpd), and the plasma membrane ATPase gene (ATP). These fungi belonged to Alternaria kordkuyana and A. rosae, which were the first detection of those taxa for the Eastern Europe. Alternaria kordkuyana was isolated from cereal seeds and eleuthero leaves. Alternaria rosae was obtained from oat seed. All strains of sect. Pseudoalternaria were not able to produce alternariol mycotoxin, as well as the majority of A. sect. Infectoriae strains.


Introduction
Alternaria Nees fungi are ubiquitous and frequently isolated from plants, soil, air, dust, and water-damaged buildings [1,2]. Many Alternaria species infect crop plants in the field and cause diseases, leading to significant economic losses [1,3,4]. This genus is characterized by dark colored, multicelled conidia with transverse and longitudinal septa. Conidia commonly occur in chains or sometimes remain solitary and usually contain an apical beak or tapering apical cells [5]. Early Alternaria taxonomy was inconsistent as it was based only on general morphological characteristics. The later taxon criteria within this genus used many morphological characteristics of conidia and a threedimensional sporulation pattern. It led to the description of several Alternaria species groups [6][7][8]. Later based on combination of morphological and multilocus phylogenetic characteristics, many Alternaria species-groups were converted into the sections, the taxa of the sub-generic rank [9]. Among them, the A. infectoria species group was distanced from other small-spored Alternaria spp. by morphological and biochemical characters and molecular markers [10][11][12]. The majority of the A. infectoria species group representatives was placed in the sect. Infectoriae. However, some A. infectoria-like strains were recently placed in the sect. Pseudoalternaria, which is strongly supported as the sister group to sect. Infectoriae [13]. Currently, this section includes at least seven species.

PCR Primer Development
The primer pair APsF (CCGCCGCCAATCCAGTTC) and APsR (AAGGTTGGTCTTCTCG-GAAG) specific for DNA of Alternaria sect. Pseudoalternaria fungi was designed based on AT-Pase gene sequences from Alternaria spp. available in the GenBank database ( Table 2). Primer design was performed using online software Primer3Plus [35]. These primers were expected to amplify a region of 424 bp.  Alternaria spp. were cultured on potato sucrose agar medium (PSA) for 7 days. The genomic DNA from the mycelium (10-50 mg per strain) was isolated using a genomic DNA purification kit (Thermo Fisher Scientific, Vilnius, Lithuania) according to the manufacturer's protocol.
The specificity of the primers APsF and APsR was analyzed by PCR with DNA, using all 66 Alternaria spp. strains. The amplification was also performed with the primers Ain3F and Ain4R that had previously been developed for the identification of the A. infectoria-like fungi [21]. Amplification was done in a 25 µL reaction mix comprising 1 × PCR-buffer with 25 mM MgCl 2 , 0.2 mM dNTP, 0.5 U Taq polimerase (all reagents Thermo Fisher Scientific, Vilnius, Lithuania), and 0.5 µM primers (Eurogen, Moscow, Russia) with a BioRad C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) using the following cycling protocol: 95 • C for 3 min, 40 cycles of 95 • C for 20 s, 65 • C for 20 s, and 72 • C for 40 s, followed by final elongation of 72 • C for 3 min. Visualization of results was performed by electrophoresis of amplification products in 1% agarose gel.

Genomic DNA Isolation, Sequencing, and Phylogenetic Analysis
The Alternaria strains whose DNA was amplified with the primers APsF and APsR were included in the phylogenetic study. The region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was amplified using primers gpd1 and gpd2 [38]. Primers ATPDF1 and ATPDR1 were used to amplify part of plasma membrane ATPase gene (ATP) [9]. Sequencing of the fragments was done on an ABI Prism 3500 sequencer (Applied Biosystems, Hitachi, Japan) using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA).
Alignment of the sequences obtained for each strain was performed using Mega X 10.1 program [39]. Basic Local Alignment Search Tool (BLAST) was used to perform similarity search, by comparing the consensus sequences with sequences in NCBI GenBank database. The closest matching sequences were added to the alignment ( Table 2). Phylogenetic analysis of combined sequences consisted of maximum likelihood (ML) and maximum parsimony (MP) performed with Mega X 10.1. ML analysis was completed on a neighbor joining starting tree, generated automatically. Nearest neighbor interchange was used as the heuristic method for tree inference. The best nucleotide substitution model used for building the ML trees (TN93 + G) was also determined in MEGA X 10.1. MP analysis was performed using the heuristic search option with 100 random taxon additions and the subtree pruning regrafting method as the branch-swapping algorithm. All characters were unordered and of equal weight, and gaps were treated as missing values. Input parameter "maxtrees" was set to 100, and branches of zero length were collapsed. Bootstrap supports values for ML and MP trees branches were calculated with 1000 replicates. Additionally, Bayesian probability (BP) calculation was done with Mr. Bayes v. 3.2.1. in Armadillo v. 1.1 [40]. using a Markov chain Monte Carlo (MCMC) sampling method. The general time-reversible model of evolution, including estimation of invariable sites and assuming a gamma distribution with six rate categories, was used for Bayesian inference analyses. Four MCMC chains were run simultaneously, starting from random trees for 1000 generations and sampled every tenth generation for a total of 10,000 trees. Sequence data was deposited in GenBank (MW478365-MW478374).

Morphology Characterization
For examination of colony morphology, the strains were grown on potato carrot agar (PCA) [15] with 12:12 h L:D photoperiod for 7 days and on PSA without lighting. For sporulation assessment, the strains were cultured on PCA at 24 • C with 12:12 h L:D photoperiod for 3-5 days [15]. Observations of conidiation were made with a SZX16 stereomicroscope and BX53 microscope (Olympus, Tokyo, Japan). Images were captured with a Prokyon camera (Jenoptik, Jena, Germany).

Toxin-Producing Ability
For analysis of toxin-producing ability, 25 Alternaria strains belonging to four sections (14 sect. Infectoriae, 5 sect. Pseudoalternaria, and 3 sects. Alternaria and Panax) were selected. The selected strain was grown in 15-mL glass vials containing 1.0 mL malt extract agar (MEA), 1 g of polished rice (rice), or pearl barley (barley) in 3-6 replicates. The moisture content of the rice and barley was adjusted by the addition of 2 mL of water before autoclaving at 120 • C for 20 min. Inoculation of MEA and groats was done by suspension of conidia (50 µL at 50 CFU/µL). The strains were cultured for 7 days at 25 • C in complete darkness.
For mycotoxin extraction, 3 mL of the mixture acetonitrile and water (84:16, v/v) was added in each vial. The vials ware shaken vigorously and left to incubate for 12-14 h. After repeated shaking and tenfold dilution with a buffer solution, the extract was used for indirect competitive enzyme-linked immunosorbent assay [41]. If necessary, 100-, 1000-or 10,000-fold dilutions of the extract were made with a buffer solution containing 10% of the acetonitrile and water mixture (84:16, v/v). Determination of AOH with a detection limit of 0.4 ng/mL was performed using certified kits and the manufacturer's protocol (VNIIVSGE, Moscow, Russia). The toxin-producing ability of the Alternaria spp. strains was determined as the AOH content in 1 g of substrate (µg/g).

Statistical Analysis
Experiments to study the toxin-producing ability of the strains were conducted twice, using at least three biological replicates. Statistical analysis of the obtained results was performed using Statistica 10.0 software. The mean values and the confidence interval were calculated at a 95% significance level.

Specificity of Primers and Strains Identification with PCR
PCR with primers APsF and APsR amplified DNA of only both representative strains of sects. Pseudoalternaria, A. arrhenatheri, and A. parvicaespitosa (Table 1). However, with primers Ain3F and Ain4R, DNA was amplified for all seven representative Alternaria sect. Infectoriae strains. Importantly, PCR with both primers sets did not amplify DNA of the 21 Alternaria spp. belonging to the eight other Alternaria sections and A. brassicae.
The newly designed primers APsF and APsR, along with Ain3F and Ain4R, were used to search for Alternaria sect. Pseudoalternaria among 35 local A. infectoria-like strains. These strains, according to the sum of morphological characters, could be representatives of both sects. Infectoriae and Pseudoalternaria. DNA from 30 Alternaria strains was amplified with primers Ain3F and Ain4R, but five other Alternaria spp. were amplified with primers APsF and APsR. The latter five strains were included in the further phylogenetic study.

Molecular Phylogeny
Adjusted and aligned gpd and ATP sequences had the lengths of 492 and 1232 bp with 82 (16.7%) and 185 (15.0%) parsimony-informative sites per genome locus, respectively. Topology of trees built by different methods was the same and also was concordant with phylogenetic relation between Alternaria species reconstructed previously [2,17,18]. The newly designed primers APsF and APsR, along with Ain3F and Ain4R, were used to search for Alternaria sect. Pseudoalternaria among 35 local A. infectoria-like strains. These strains, according to the sum of morphological characters, could be representatives of both sects. Infectoriae and Pseudoalternaria. DNA from 30 Alternaria strains was amplified with primers Ain3F and Ain4R, but five other Alternaria spp. were amplified with primers APsF and APsR. The latter five strains were included in the further phylogenetic study.

Molecular Phylogeny
Adjusted and aligned gpd and ATP sequences had the lengths of 492 and 1232 bp with 82 (16.7%) and 185 (15.0%) parsimony-informative sites per genome locus, respectively. Topology of trees built by different methods was the same and also was concordant with phylogenetic relation between Alternaria species reconstructed previously [2,17,18].

Alternaria Sect. Pseudoalternaria Morphology
Colonies of A. kordkuyana MF P094121 on PSA were 79 mm diam. after 7 days without lighting, flat, felty, light gray at center, and white at edge, with light taupe reverse; on PCA, 79 mm diam. after 7 days with 12:12 h L:D photoperiod, flat, velvet, with poor aerial mycelium, light brown on top, and reverse ( Figure 2C). Conidia were ellipsoidal, obclavate, smooth, or verrucose, usually with darkened and constricted median transeptum, 9-34 × 6-12 µm (av. 20 × 9 µm), mainly with secondary conidiophores from apical cell 4-22 × 4-5 µm (av. 10 × 4 µm) (Figure 2A). Conidia were formed in simple or branched chains up to five units in a row ( Figure 2B). Sporulation clamps consisted initially of 2-7 conidia or up to 15 (20) conidia in the old cultures. Branching of the chains occurred mainly due to the presence of 2-4 conidiogenous loci on the apical secondary conidiophores. Lateral secondary conidiophores were not observed. In general, the three-dimensional sporulation pattern was similar to that of sect. Infectoriae but differed by shorter chains and smaller conidial clumps.
Colonies of A. rosae MF P457041 on PSA were 71 mm diam., flat, felty, dark gray at center, and white at edge, with dark brown reverse, turning light brown towards the edge; on PCA, 76 mm diam., flat, cottony, white, and taupe in center and light brown at edge, with light brown reverse ( Figure 2D). This strain in our study remained sterile on PCA and PSA under the used temperature and lighting conditions.

Alternariol-Producing Ability
Only four of the 25 Alternaria spp. strains analyzed produced AOH with concentrations ranging from 1 to 1910 μg/g, depending on the substrate (Table 1). Of these, three strains of Alternaria sect. Alternaria, as well as MF P457121 in sect. Infectoriae, were AOH-producing. The remainder of the representatives of sect. Infectoriae (13 strains), all strains belonging to sect. Pseudoalternaria (5 strains) and Panax (3 strains), did not produce the AOH on the analyzed substrates in detectable amounts. Colonies of A. rosae MF P457041 on PSA were 71 mm diam., flat, felty, dark gray at center, and white at edge, with dark brown reverse, turning light brown towards the edge; on PCA, 76 mm diam., flat, cottony, white, and taupe in center and light brown at edge, with light brown reverse ( Figure 2D). This strain in our study remained sterile on PCA and PSA under the used temperature and lighting conditions.

Alternariol-Producing Ability
Only four of the 25 Alternaria spp. strains analyzed produced AOH with concentrations ranging from 1 to 1910 µg/g, depending on the substrate (Table 1). Of these, three strains of Alternaria sect. Alternaria, as well as MF P457121 in sect. Infectoriae, were AOH-producing. The remainder of the representatives of sect. Infectoriae (13 strains), all strains belonging to sect. Pseudoalternaria (5 strains) and Panax (3 strains), did not produce the AOH on the analyzed substrates in detectable amounts.

Discussion
Accurate identification of Alternaria spp. is complicated and it is often only possible using molecular methods [2]. The phylogenetic approach to differentiation of Alternaria spp. requires the analysis of sequences of several loci [2], while specific PCR is simple and convenient when primer specificity is sufficiently precise. A number of primers specific for Alternaria were designed. There are primers for identification of Alternaria fungi at the genus level [42]. Primer sets intended for identification of Alternaria alternata-like fungi [43][44][45] have specificity that is limited to sect. Alternaria. One of these primer sets was successfully used for quantitative detection of sect. Alternaria fungi in cereals grain [46].
The primers Ain3F and Ain4R were developed for the identification of the A. infectoria-like fungi [21]. Their specificity for sect. Infectoriae was re-examined and confirmed in this study. The present study designed and validated the first primer set (APsF and APsR) specific for Alternaria sect. Pseudoalternaria fungi. These primers, in combination with primer sets for sects. Alternaria and Infectoriae, can be used for analysis of mycobiota of grain or other agricultural or clinical samples. Sometimes, grain samples with high Alternaria infection levels should be assessed for their potential toxicity. With these three primer sets, PCR can be used for this purpose as a simple and quick method.
In the present study, of 35 Eastern European A. infectoria-like strains, five strains (14.3%) were found to belong to sect. Preudoalternaria. All of these stains came from Leningrad and Belgorod Regions of European Russia. Despite the fact that A. sect. Pseudoalternaria fungi have previously been found on common crops, wheat [17,18], apple [47], and napa cabbage [20], no A. sect. Pseudoalternaria fungi have been detected in Eastern Europe. The present study is the first to establish the presence of A. sect. Pseudoalternaria species in Russia.
Four local strains were identified as A. kordkuyana and were isolated from wheat and oat grain (Poaceae) and eleuthero leaves (Siberian ginseng, Araliaceae) in central and northwestern European Russia. Previously, A. kordkuyana was isolated from wheat plant in Iran [17], from apple in Chili [47], and from herbivore dung in Spain [19]. In earlier unpublished work [48], a group of six short-chained A. infectoria-like strains from poaceous plants were found. All strains had ITS region sequences that differed from genuine A. infectoria species group strains. Five of them have identical sequences, and one of those strains, MF P457051, still exists in the collection and was used in the present study. Thus, with a high degree of confidence, we can report that A. kordkuyana was also found on Triticum durum in the Omsk Region (western Siberia) and Hordeum vulgare in the suburbs of St. Petersburg. Another strain of an unidentified species of A. sect. Pseudoalternaria was isolated from Leymus arenarius in the Murmansk Region (Northwestern European Russia).
One local strain isolated from oat grain in the present study was presumptively identified as A. rosae. Information on the distribution of this species is fragmentary. Initially, A. rosae was isolated from stem lesions of sweet briar (Rosa rubiginosa, Rosaceae) in New Zealand and described by E.G. Simmons [15]. Additionally, this species was isolated from the roots of Arabidopsis thaliana (Brassicaceae) in Germany and its full genome was sequenced [49].
Apparently, A. kordkuyana and other A. sect. Preudoalternaria species are cosmopolitan and not confined to one species or family of host plants. Existing information indicates a potentially wide distribution but low occurrence of these fungi in the mycobiota of crops.
Alternaria kordkuyana and A. rosae strains had similar growth rate. The diameter of the colonies was similar to that of previously studied strains of A. kordkuyana [17] and A. rosae [15]. Conidia of A. kordkuyana MF P094121 were average 20 × 9 µm, mainly with oblong secondary conidiophores from apical cell av. 10 × 4 µm. According Poursafar et al. [17], conidia of this species were 15-50 × 7-12 µm, with secondary conidiophores arising from the apical cell, which may be up to 20 µm long.
Perhaps Alternaria fungi are abundant in the grain mycobiota of all cereal-producing countries [50][51][52][53], including Russia [46,53]. Alternaria sect. Alternaria fungi are the most frequent group among Alternaria spp. in grain [34,46,54,55] and are also widespread on other plants [1,56]. Alternaria sect. Infectoriae are also abundant in cereal grain and have often been reported to be on poaceous plants [10,11,17,57], but were occasionally detected on other hosts [11,58,59]. In the present study, strains of both sections were also mainly isolated from cereal plants. Presumably, A. sect. Pseudoalternaria fungi is also common in cereals, but when identified by morphological characters, may be misidentified as Alternaria species from sect. Infectoriae. In addition, A. sect. Panax fungi were identified on cereals [60][61][62] and may add confusion to species identification.
Alternaria fungi are producers of various mycotoxins [63]; their presence affects the quality and safety of grain-based food and feed [26,64]. Among Alternaria mycotoxins, AOH has genotoxic, mutagenic, and carcinogenic effects on humans and animals and is one of the most frequently analyzed toxins [24]. A draft EU Commission Recommendation on the monitoring of Alternaria mycotoxins in food was issued: the benchmark value of AOH in cereal-based foods for infants and children was 5 µg/kg [65].
This mycotoxin was detected in about 20% of samples of wheat, barley, and oats grain grown in Russia in 2009-2019, and its amounts varied from 5 to 675 µg/kg [66]. In another study, the analysis of grain samples grown in the Urals and West Siberia in 2017-2019 revealed a similar occurrence of AOH (27%) but smaller concentrations of this mycotoxin (2-53 µg/kg) [46]. The unequal distribution of occurrence and amounts of AOH in grain samples from different regions was noted [66]. Additionally, AOH was detected in grain samples from Europe [33,67], Asia [27,34], North and South America [68,69], and Australia [51].
Alternaria sect. Alternaria fungi are well known as producers of AOH [10,12,33,55]. In the present study, when cultured on the grain substrate (rice and pearl barley), three A. sect. Alternaria strains produced 2.5-4.8 times higher amounts of AOH than on MEA. The substantial effect of substrate on the intensity of AOH production is well known [70][71][72].
In Central and North Italy, the production AOH by A. sect. Alternaria strains isolated from wheat grain was determined to range from 1 to 5620 mg/kg and 1-8064 µg/g, respectively [33,55]. However, some A. sect. Alternaria strains did not produce AOH in vitro: 16% of strains (129 in total) did not produce mycotoxins [12]. However, it can be presumed that AOH-lacking strains were incorrectly identified and perhaps actually belonged to other sections.
Information on the ability of A. sect. Infectoriae to produce AOH is inconsistent. Previously, an analysis of 10 A. infectoria strains did not demonstrate any ability of these strains to produce AOH on nine different types of agar media [73]. Additionally, none of the 20 A. infectoria strains isolated from food crops in Argentina produced AOH when cultured on DRYES agar medium [74]. In contrast, 75% of A. infectoria strains from Italy produced AOH up to 223 µg/g when cultured on rice [33]. According to Oviedo et al. [12], 81% of A. infectoria strains analyzed produced AOH when cultured on ground rice-corn steep liquor medium at concentrations of 1.8-433 µg/g. In the study by Ramires et al. [55], all A. infectoria strains analyzed produced AOH in the range 0.3-20 mg/kg. The ability to produce AOH varies greatly between strains and geographic populations of A. sect. Infectoriae fungi. It is important to remember that precise species identification in this section is complicated and often A. infectoria is used in a broad sense and should be readily assumed to be an Alternaria sp. sect. Infectoriae.
In the present study, only one strain, MF P457121, of 14 A. sect. Infectoriae strains, produced AOH, and that was at a concentration 8-70 times less than in the average A. sect. Alternaria strains cultured on the same substrates. Additionally, when cultured on rice and pearl barley, the amount of AOH produced by MF P457121 was 10-20 times higher than on MEA. Previously, the production of AOH by A. sect. Alternaria strains (A. alternata, A. arborescens, and A. tenuissima) was 95 times higher than that by A. infectoria strains on a rice substrate [75].
None of the five Alternaria spp. from sect. Pseudoalternaria strains analyzed produced AOH when cultured on the various substrates tested. Currently, the toxigenicity of only one A. sect. Pseudoalternaria strain isolated from wheat grain has been reported [54], with the strain ITEM 17904 producing AOH on rice medium at a low concentration (1.5 mg/kg). There is no available information on the toxigenicity of A. avenicola or other Alternaria spp. from sect. Panax. In the present study, none of the three strains in sect. Panax produced AOH.
Therefore, Alternaria spp. strains from sects. Infectoriae and Pseudoalternaria have the ability to produce AOH. However, the contribution of these fungi to the contamination of grain with AOH, and possibly many other more dangerous mycotoxins, has not been adequately studied compared to that of A. sect. Alternaria fungi, as these are more abundant and have AOH production potential 2-3 orders of magnitude higher.