# Distribution of large-spored Alternaria species associated with early blight of potato and tomato in Algeria

Djida Ayad, Djihad Aribi, Bruno Hamon, Abdelaziz Kedad, Philippe

Simoneau, Zouazi Bouznad

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Djida Ayad, Djihad Aribi, Bruno Hamon, Abdelaziz Kedad, Philippe Simoneau, et al.. Distribution of large-spored Alternaria species associated with early blight of potato and tomato in Algeria. Phytopathologia Mediterranea, 2019, 58 (1), pp.139-149. 10.13128/Phytopathol_Mediterr-23988. hal-02622664

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**Citation:** Ayad D., Aribi D., Hamon B., Kedad A., Simoneau P., Bouznad Z. (2019) Distribution of large-spored *Alternaria* species associated with early blight of potato and tomato in Algeria. *Phytopathologia Mediterranea* 58(1): 139-149. doi: 10.13128/Phytopathol_Mediterr-23988

**Accepted:** November 19, 2018

**Published:** May 15, 2019

**Copyright:** © 2019 Ayad D., Aribi D., Hamon B., Kedad A., Simoneau P., Bouznad Z. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper and its Supporting Information files.

**Competing Interests:** The Author(s) declare(s) no conflict of interest.

**Editor:** Vladimiro Guarnaccia, University of Torino, Italy.

Research Papers

# Distribution of large-spored *Alternaria* species associated with early blight of potato and tomato in Algeria

DJIDA AYAD<fnref n="1" />,<fnref n="2" />,*, DJIHAD ARIBI<fnref n="1" />, BRUNO HAMON<fnref n="2" />, ABDELAZIZ KEDAD<fnref n="1" />, PHILIPPE SIMONEAU<fnref n="2" />, ZOUAOUI BOUZNAD<fnref n="1" />

**Abstract.** Potato and tomato are important crops in Algerian agriculture, and both are threatened by abiotic and biotic stresses, and early blight is a major disease affecting both crops. Surveys carried out from 2012 to 2015 in 12 major growing regions for these crops yielded a total of 247 *Alternaria* isolates having morphological and cultural characteristics of sections *Alternaria* and *Porri*. Since early blight symptoms and morphological characteristics of the isolates did not allow sharp distinction between the different large-spored species of *Alternaria*, the isolates in section *Porri*, often considered primary causes of the diseases, were selected for molecular characterization by diagnostic PCR using specific primers. This allowed species identification of 147 *Alternaria* isolates as *A. solani*, *A. protenta*, *A. grandis* or *A. linariae*. These species were present on potato and tomato crops at varying frequencies, depending on the hosts and on bioclimatic locations. Pathogenicity tests for the four species, on detached leaflets and whole seedlings, showed that all were pathogenic to potato and tomato, with varying virulence. These results suggest that parasitic specialization of these *Alternaria* species on solanaceous plants should be reconsidered.

**Keywords.** Solanaceous crops, parasitic specialization, prevalence.

## INTRODUCTION

Potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.) are important crops grown in Algeria. They are distributed differently in the various bioclimatic zones of the country, and are grown with different cultural practices. Potato crops occupy significant area in several regions of Algeria, from north (Ain Defla) to south (El Oued), and from east (Skikda, Guelma) to west (Mostaganem, Mascara). Tomato, however, is mainly cultivated in the northern part of the country, less extensively and mostly in plastic houses, as in the Biskra region. Both crops are often cultivated side by side or follow one another in rotations for several years, in many coastal regions of the

<footnote n="1">¹ENSA-El Harrach, Laboratoire de Phytopathologie et Biologie Moléculaire. Algiers, Algeria</footnote>

<footnote n="2">²UMR 1345 IRHS, Université d'Angers-INRA-Agrocampus Ouest, SFR QUASAV, 42 Rue G. Morel, F-49071 Beaucouzé, France</footnote>

<footnote>*Corresponding author: medaydji@gmail.com</footnote>
country. Intensification of these two crops without com-
pliance to good agricultural practices led to the develop-
ment of important diseases caused by fungi and oomy-
cetes, including early blight and late blight.

The first reference mentioning *A. solani* Sorauer (= *Macrosporium solani* Ellis & G. Martin) as a pathogen causing potato leaf blight was that of Galloway (1891) in Australia. Chester (1892) then reported this pathogen in the United States of America, other solanaceous cultivat-ed plants (Van der Waals *et al.*, 2001; Odilbekov, 2015). A few years later, *A. solani* and *A. alternata* (Fr.) Keissl. were identified on potato leaves but *A. alternata* was considered first as saprophytic (Jones and Grout, 1897). It was not until 1984 that *A. alternata* was recognized as pathogenic on several solanaceous plants (Droby *et al.* 1984). The name “early blight” for the disease caused by *A. solani* was coined by Jones (1893) to distinguish this from late blight. The symptoms of the early blight appear on the leaves as dark, elongated or circular lesions each with concentric rings surrounded by a yellow halo.

On the basis of cultural techniques and morphologi-
cal characteristics of the conidia (size and beak length),
Simmons (2000, 2007) described several new *Alternaria*
species on *Solanaceae*, that may be responsible for ear-
ly blight on potato and tomato. Thus, new species were
described, which are morphologically very similar to
*A. solani*. These include *A. grandis* E.G. Simmons on *S.
tuberosum*, *A. tomatophila* E.G. Simmons on *S. lycopers-
cicum*, *A. cretica* E.G. Simmons & Vakal. On *S. lycopersi-
cum* var. *esculentum*, and *A. subcylindrica* E.G. Simmons
& R.G. Roberts on *S. lycopersicum* var. *cerasiforme*.

More recently, molecular and genomics techniques applied to *Alternaria* allowed redefinition of various species with large conidia commonly isolated from *Solanaceae*, and considered as the primary causes of early blight. Woudenberg *et al.* (2014), by synonymizing *A. linariae* (Neerg.) E.G. Simmons with *A. cretica* E.G. Simmons & Vakal., *A. cucumericola* E.G. Simmons & C.F. Hill, *A. subcylindrica*, *A. tabasco* E.G. Simmons & R.G. Roberts, and *A. tomatophila*, have expanded the host range of *A. linariae* to *Solanaceae*, *Cucurbitaceae*, and *Scrophulariaceae*. *Alternaria linariae*, a common pathogen of tomato, was also reported on potato during surveys carried out in Algeria (Ayad *et al.*, 2018). *Alternaria Grandis*, which was confused for a long time with *A. solani*, has been regularly reported in many countries on potato (Lourenço *et al.*, 2009; Cardoso, 2014; Bessadat *et al.*, 2016; Landschoot *et al.*, 2017). *Alternaria grandis* was also recently isolated from tomato (Bessadat *et al.*, 2017) in the northwest of Algeria. *Alternaria protenta*, which is closely related to *A. solani* and whose host range was extended to *Asteraceae*, *Euphorbiaceae*, *Gramineae* and

*Solanaceae* by Woudenberg *et al.* (2014), was recently
detected on potato in Belgium (Landschoot *et al*., 2017)
and in Algeria (Ayad *et al*., 2017).

The aims of the present study were, firstly, to identi-
fy at the species level the large-spored *Alternaria* isolates
obtained from surveys carried out during three crop-
ping years on different potato and tomato fields across
the main bioclimatic areas of Algeria. Since early blight
symptoms and the morphological characteristics of the
isolates do not allow distinction between the different
large-spored *Alternaria* spp., specific identification of the
isolates was based on PCR techniques. The second objec-
tive was to assess the aggressiveness of isolates on their
respective potato and tomato hosts, and to clarify the
parasitic specialization of replace by large-spored *Alter-
naria* species by artificial cross inoculations *in vitro* and
under conditions similar to those occurring in the field.

MATERIALS AND METHODS

Surveys and sampling

Surveys were carried in 60 locations across 12
regions in Algeria, with different cropping systems
(fields and greenhouses). Three to four plots were
sampled in each region during three successive crop-
ping years: 2012-2013, 2013-2014 and 2014-2015. The
majority of the localities were selected from the main
potato- and tomato-producing regions represented by
different bioclimatic areas of Algeria: Mediterranean
climate (Algiers, Tipaza, Guelma and Skikda), semi-
arid climate (Mostaganem, Chlef, Mascara, Ain Defla
and Bouira), and arid climate (Laghouat, Biskra and El
Oued) (Figure 1). Sampling was carried out from pota-
to and tomato plant organs showing the typical symp-
toms of early blight: 164 isolates were collected from
potato leaves essentially and 83 from leaves, stems and
fruits of tomato.

Isolation, purification and conservation of isolates

Isolations were performed according to the method
of Van der Waals et al. (2004). Small tissue pieces (3 to
4 mm²) were cut from individual lesion edges, disinfect-
ed in 1% active NaOCl for 3 min, washed twice in ster-
ile distilled water, and then dried with absorbent sterile
paper. Four pieces of tissues were plated on individual
Petri plates containing Potato Dextrose Agar (PDA),
and these were incubated at 22°C under continuous
light for 1 to 2 weeks. The purification of the Alter-
naria spp. isolates as single conidium cultures was car-
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Geographic localities in the different bioclimatic areas: 1 = Mediterranean climate, 2 = semi-arid climate, 3 = arid climate</figcaption></figure>

ried out between the 3rd and the 5th day of incubation. Plates containing the single conidium cultures were then placed under near-UV (12h dark and 12h light) to induce sporulation and allow further morphological characterization.

Single conidium cultures for short-term use were grown in Petri dishes containing a PDA for 12 d then stored at 4°C. For medium term use, 12-d-old PDA cultures in test tubes were stored at 4°C. For long term use, mycelium/agar plugs (8 mm diam.) were stored at 80°C in cryotubes containing sterile glycerol (30% w/w).

*Identification of the large-spored *Alternaria* species using molecular markers*

On the basis of the cultural and the morphological characteristics, 155 isolates with large conidia (out of a total 247 isolates) were selected for molecular characterization by PCR using specific primers, for analysis of genomic DNA by PCR/RFLP, using double enzymatic digestion of a portion of the calmodulin gene and sequencing the calmodulin and the RPB2 genes. Isolates were grown on PDA medium at room temperature for 10 d. Total genomic DNA from fungal samples was extracted according to the method described by Goodwin and Lee (1993). The DNA extracts of the 155 isolates were amplified using two sets of specific primer pairs to differentiate between *A. linariae* and *A. solani* and related species (*A. grandis* and *A. protenta*) (Gannibal *et al.*, 2014). The primer pair *OasF7* and *OasR6* amplified a 164 bp fragment from the *Alt a1* gene of *A. solani*, *A. grandis* and *A. protenta*, and the primer pair *OatF4* and *OatR2* amplified a 438 bp fragment from the calmodulin encoding gene of *A. linareae*. The PCR conditions were as described by Gannibal *et al.* (2014). To differentiate between *A. grandis* and *A. solani sensu lato* (i.e. includ-

ing *A. protenta*), a portion of the calmodulin gene was amplified from the isolates that each gave a positive signal with the primer pair *OasF7* – *OasR6* using the primer pair *CALDF1* / *CALDR1* (Lawrence *et al.*, 2013). The resulting PCR products were double digested with the restriction enzymes *RsaI* and *HaeII*. The digestion products were then separated by electrophoresis in 2% agarose gels and visualized under UV light after staining with ethidium bromide. Predicted restriction patterns mainly differed by the size of the larger fragment, i.e. 420 bp for *A. solani sensu lato* and 292 bp for *A. grandis* (Figure 2). Identification of the *Alternaria* species was validated by sequencing the fragment of the calmodulin locus amplified as described above from 42 isolates. To confirm the presence of *A. protenta*, a portion of the RPB2 locus was amplified from the isolates identified as *A. solani* based on their calmodulin sequence, using the primer pair RPB2-5F2 (Sung *et al.*, 2007) and RPB2-7cR (Liu *et al.*, 1999), and the resulting PCR product was sequenced. Sanger sequencing was performed by GATC Biotech. Sequence analyses were carried out using the Phylogeny.fr web service (Dereeper *et al.*, 2008). Multiple sequence alignments were generated with MUSCLE and curated using the Gblocks algorithm. Maximum likelihood (ML) analyses were performed with PhyML. The robustness of the ML topologies was evaluated using the Shimodaira-Hasegawa (SH)-like test for branches.

*Pathogenicity tests of large-spored *Alternaria* species on their respective hosts*

To confirm pathogenicity of isolates, inoculations onto detached leaflets and whole plants were performed on susceptible varieties of tomato (cv. Marmande) and

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Typical restriction patterns obtained by double digestion of PCR product corresponding to a portion of the calmodulin gene from *A. solani* and *A. grandis*. Size of the larger fragment, 420 bp for *A. solani sensu lato* (S) and 292 bp for *A. grandis* (G)</figcaption></figure>
potato (cv. Spunta). These isolates were selected for their
ability to produce conidia. Conidium suspensions were
prepared from 15-d-old cultures. The final concentration
of the suspensions was adjusted to 10⁴ conidia mL⁻¹. The
inoculations were performed by depositing drops (20 µL
each) on detached leaflets or by spraying suspensions
onto whole plants. Disease severity was evaluated at 15
d-post-inoculation, using a visual rating scale from 1 to
9 expressing the extension of necrosis CIP (International
Potato Center) (2008).

RESULTS

*Characterization of the large-spored *Alternaria* species according to early blight symptoms on potato and tomato*

Isolation from plant samples showing typical early
blight symptoms yielded 247 isolates (164 from potato
and 83 from tomato). On the basis of the cultural and
morphological characteristics of the colonies and conid-
ia, the isolates were divided in three groups. The first
(Group A: 92 isolates) included all the isolates with abun-
dant conidium production and small catenulate conidia
and identified as species belonging to section *Alternaria*.
The second (Group B: 92 isolates) contained all isolates
producing large solitary beaked conidia, related to spe-
cies belonging to the section *Porri*. The third (Group C:
63 isolates) consisted of large-spored isolates that only
formed sterile mycelium after culture purification. In
relation to the host plants, 89% of the isolates belong-
ing to the section *Porri* originated from potato, and 73%
of the non-sporulating isolates originated from tomato.
No correlation was found between the presence of yel-
low halos on symptomatic leaves and the production of
a diffusible pigment on growth medium by the isolates
from the diseased samples. Similarly, the various forms
of symptoms generated colonies producing either large or
small conidia. Thus, there was no relationships between
necrosis size (small, medium or large necrotic lesions),
the types of the colonies in culture medium (sporulating
or non-sporulating) and the conidium types (large soli-
tary or small catenulate conidia).

*Molecular characterization and identification of large-
spored *Alternaria* species*

Given the very similar morphological characteris-
tics of the conidia, and the difficulty to distinguish the
different *Alternaria* species within the section *Porri*,
molecular markers and DNA-sequencing were used for
their identification. The small-spored isolates (Group

A) were not included in this examination. The three
molecular approaches allowed clear species identifica-
tion for 147 of 155 isolates from Groups B and C, while
eight isolates could not be identified due to problems in
DNA extraction and amplification at the selected loci
(Table 1). Preliminary tests were carried out for the
identification of the 147 isolates using two pairs of spe-
cific primers that differentiate *A. solani*, *A. protenta* and
*A. grandis* (OasF7/OasR6) from *A. linariae* (OATF4/
OATR2). All the tested isolates generated positive sig-
nals with one of the two primer pairs, confirming the
presence of these species on potato and tomato crops
in Algeria. In this analysis, 37 isolates including seven
from potato and 30 from tomato, were identified as *A.
linariae* by amplification of the 438 pb from the gene
encoding for calmodulin (Table 1). A PCR product cor-
responding to a fragment of the Alt a1 gene (164 bp)
was amplified from the remaining 110 isolates. These
isolates, of which 86 were isolated from potato and 24
from tomato, could be assigned to either *A. solani* or
to phylogenetically related-species, i.e. *A. grandis* and
*A. protenta*. Further identification was conducted in
order to differentiate between *A. solani* and *A. grandis*
isolates using PCR/RFLP, by amplifying the calmodulin
locus followed by restriction enzyme digestion (*Hae*II
and *Rsa*I). Based on the characteristic electrophoretic
profiles of the restricted PCR products, *A. solani* and *A.
protenta* were separated from *A. grandis*. Thus, of the
110 isolates that gave positive signals with the OasF7/
OasR6 primer pair, 93 were *A. solani*, 12 were *A. gran-
dis* and five showed abnormal enzymatic restriction
digestion patterns that may be due to partial diges-
tion with at least one of the restriction enzymes. These
five isolates, as well as 37 other representative isolates
obtained from the two host plants collected in nine of
the twelve surveyed Algerian regions and represent-
ing the three identified species (*A. linariae*, *A. grandis*
and *A. solani*), were selected for species confirmation
by DNA sequencing which was firstly performed at the
calmodulin locus. Phylogenetic analyses of sequences
derived from the calmodulin locus of the 42 isolates
(Figure 3) separated the isolates into two clades: the
first (A) contained the isolates previously identified by
PCR with specific primers as *A. linariae*, and the sec-
ond (B) included two sub clades (B1 and B2) regrouping
isolates previously identified as *A. grandis* (sub clade
B1) and *A. solani* (sub clade B2). DNA sequencing at the
RPB2 locus then carried out on the isolates of the sub
clade B2 gave the phylogenetic tree shown in Figure 4,
in which among the 17 isolates of *A. solani*, three were
identified as *A. protenta*, two of which were isolated
from the potato and one from tomato.
<table><caption>**Table 1.** Identification of large-spored of *Alternaria* spp isolates.</caption><thead><tr><th>Isolate</th><th>Species<sup>a</sup></th><th>Host</th><th>Geographic origin</th><th>PCR OATF4/ OATR2</th><th>PCR OAsF7/OAsR6</th><th>PCR RFLP Profile<sup>b</sup></th><th>CAL</th><th>Rpb2</th></tr></thead><tbody><tr><td>DA001</td><td><strong>A. linariae</strong></td><td>Potato</td><td>Tipaza</td><td>+</td><td>-</td><td>ND</td><td>MH243795</td><td>-</td></tr><tr><td>DA002</td><td>A. linariae</td><td>Potato</td><td>Tipaza</td><td>+</td><td>-</td><td>ND</td><td>MH243769</td><td>-</td></tr><tr><td>DA003</td><td><strong>A. linariae</strong></td><td>Potato</td><td>Tipaza</td><td>+</td><td>-</td><td>ND</td><td>MH243793</td><td>-</td></tr><tr><td>DA005</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA006</td><td><strong>A. linariae</strong></td><td>Potato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>MH243789</td><td>-</td></tr><tr><td>DA007</td><td><strong>A. linariae</strong></td><td>Potato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>MH243794</td><td>-</td></tr><tr><td>DA008</td><td><strong>A. solani</strong></td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>MH243805</td><td>MH243818</td></tr><tr><td>DA009</td><td><strong>A. grandis</strong></td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>?</td><td>MH243790</td><td>-</td></tr><tr><td>DA010</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA011</td><td><strong>A. solani</strong></td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>MH243806</td><td>MH243820</td></tr><tr><td>DA012</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA013</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA014</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>MH243796</td><td>-</td></tr><tr><td>DA015</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA016</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA017</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA018</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA019</td><td><strong>A. solani</strong></td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>MH243808</td><td>MH243822</td></tr><tr><td>DA020</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA021</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA022</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA025</td><td><strong>A. solani</strong></td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>MH243786</td><td>MH243823</td></tr><tr><td>DA026</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA028</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA029</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA030</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA031</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA032</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA033</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA034</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA035</td><td>A. solani s.l</td><td>Potato</td><td>Mascara</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA036</td><td>A. solani s.l</td><td>Potato</td><td>Mascara</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA037</td><td>A. linariae</td><td>Tomato</td><td>Mostaganem</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA038</td><td><strong>A. grandis</strong></td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>G</td><td>MH243770</td><td>-</td></tr><tr><td>DA039</td><td>A. linariae</td><td>Potato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA040</td><td><strong>A. linariae</strong></td><td>Tomato</td><td>Mascara</td><td>+</td><td>-</td><td>ND</td><td>MH243792</td><td>-</td></tr><tr><td>DA041</td><td>A. linariae</td><td>Tomato</td><td>Mostaganem</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA042</td><td>A. linariae</td><td>Tomato</td><td>Mostaganem</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA043</td><td>A. solani s.l</td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA045</td><td>A. solani s.l</td><td>Potato</td><td>Mascara</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA046</td><td>A. solani s.l</td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA047</td><td><strong>A. grandis</strong></td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>G</td><td>MH243771</td><td>-</td></tr><tr><td>DA048</td><td>A. solani s.l</td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA049</td><td>A. solani s.l</td><td>Potato</td><td>Chlef</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA050</td><td><strong>A. solani</strong></td><td>Potato</td><td>Chlef</td><td>-</td><td>+</td><td>S</td><td>MH243772</td><td>MH243809</td></tr><tr><td>DA051</td><td><strong>A. grandis</strong></td><td>Potato</td><td>Chlef</td><td>-</td><td>+</td><td>G</td><td>-</td><td>-</td></tr><tr><td>DA052</td><td><strong>A. grandis</strong></td><td>Potato</td><td>Chlef</td><td>-</td><td>+</td><td>G</td><td>MH243773</td><td>-</td></tr></tbody></table>

(Continued)
<table><caption>**Table 1. (Continued).**</caption><thead><tr><th>Isolate</th><th>Species<sup>a</sup></th><th>Host</th><th>Geographic origin</th><th>PCR OATF4/ OATR2</th><th>PCR OAsF7/OAsR6</th><th>PCR RFLP Profile<sup>b</sup></th><th>CAL</th><th>Rpb2</th></tr></thead><tbody><tr><td>DA053</td><td>A. solani s.l</td><td>Potato</td><td>Chlef</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA054</td><td><b>A. grandis</b></td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>G</td><td>MH243774</td><td>-</td></tr><tr><td>DA055</td><td><b>A. grandis</b></td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>G</td><td>-</td><td>-</td></tr><tr><td>DA056</td><td>A. solani s.l</td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA057</td><td><b>A. solani</b></td><td>Potato</td><td>Mascara</td><td>-</td><td>+</td><td>?</td><td>MH243791</td><td>MH243819</td></tr><tr><td>DA058</td><td>A. solani s.l</td><td>Potato</td><td>Mascara</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA059</td><td>A. solani s.l</td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA060</td><td><b>A. grandis</b></td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>G</td><td>MH243775</td><td>-</td></tr><tr><td>DA061</td><td><b>A. grandis</b></td><td>Potato</td><td>Mostaganem</td><td>-</td><td>+</td><td>G</td><td>-</td><td>-</td></tr><tr><td>DA062</td><td><b>A. protenta</b></td><td>Tomato</td><td>Mostaganem</td><td>-</td><td>+</td><td>S</td><td>MH243797</td><td>MH243810</td></tr><tr><td>DA063</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA064</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA065</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA066</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA067</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA068</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA069</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA070</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA071</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA072</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA073</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA074</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA075</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA076</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA077</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA078</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA079</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA080</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA081</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA082</td><td><b>A. protenta</b></td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>?</td><td>KX870505</td><td>KX870507</td></tr><tr><td>DA083</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA084</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA085</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA086</td><td><b>A. protenta</b></td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>KX870506</td><td>KX870508</td></tr><tr><td>DA087</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA088</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA089</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA090</td><td><b>A. solani</b></td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>MH243776</td><td>MH243811</td></tr><tr><td>DA091</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA092</td><td>A. solani s.l</td><td>Potato</td><td>El Oued</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA093</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA094</td><td><b>A. solani</b></td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>MH243798</td><td>MH243812</td></tr><tr><td>DA096</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA097</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA098</td><td>A. solani s.l</td><td>Potato</td><td>Alger</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA099</td><td><b>A. grandis</b></td><td>Tomato</td><td>Alger</td><td>-</td><td>+</td><td>G</td><td>MH243777</td><td>-</td></tr><tr><td>DA100</td><td><b>A. linariae</b></td><td>Tomato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>MH243807</td><td>-</td></tr></tbody></table>

(Continued)
<table><caption>**Table 1. (Continued).**</caption><thead><tr><th>Isolate</th><th>Species<sup>a</sup></th><th>Host</th><th>Geographic origin</th><th>PCR OATF4/ OATR2</th><th>PCR OAsF7/OAsR6</th><th>PCR RFLP Profile<sup>b</sup></th><th>CAL</th><th>Rpb2</th></tr></thead><tbody><tr><td>DA101</td><td><strong>A. linariae</strong></td><td>Tomato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>MH243778</td><td>-</td></tr><tr><td>DA102</td><td>A. linariae</td><td>Tomato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA103</td><td>A. linariae</td><td>Tomato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA104</td><td>A. linariae</td><td>Tomato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA107</td><td>A. linariae</td><td>Tomato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA108</td><td>A. solani s.l</td><td>Potato</td><td>Guelma</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA109</td><td><strong>A. linariae</strong></td><td>Tomato</td><td>Tipaza</td><td>+</td><td>-</td><td>ND</td><td>MH243779</td><td>-</td></tr><tr><td>DA110</td><td>A. linariae</td><td>Tomato</td><td>Tipaza</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA111</td><td><strong>A. linariae</strong></td><td>Tomato</td><td>Tipaza</td><td>+</td><td>-</td><td>ND</td><td>MH243780</td><td>-</td></tr><tr><td>DA112</td><td>A. linariae</td><td>Potato</td><td>Skikda</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA113</td><td><strong>A. solani</strong></td><td>Tomato</td><td>Guelma</td><td>-</td><td>+</td><td>S</td><td>MH243799</td><td>MH243821</td></tr><tr><td>DA114</td><td><strong>A. solani</strong></td><td>Potato</td><td>Guelma</td><td>-</td><td>+</td><td>?</td><td>MH243788</td><td>MH243813</td></tr><tr><td>DA115</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA116</td><td>A. solani s.l</td><td>Potato</td><td>Bouira</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA117</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA118</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA119</td><td><strong>A. linariae</strong></td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>MH243800</td><td>-</td></tr><tr><td>DA120</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA121</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA122</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA123</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA124</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA125</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA126</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA127</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA128</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA129</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA130</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA131</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA132</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA133</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA134</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA135</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA136</td><td><strong>A. solani</strong></td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>MH243801</td><td>MH243814</td></tr><tr><td>DA137</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA138</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA139</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA140</td><td><strong>A. solani</strong></td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>MH243802</td><td>MH243815</td></tr><tr><td>DA141</td><td><strong>A. solani</strong></td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>MH243803</td><td>MH243816</td></tr><tr><td>DA142</td><td><strong>A. solani</strong></td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>MH243804</td><td>MH243817</td></tr><tr><td>DA143</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA144</td><td>A. linariae</td><td>Tomato</td><td>Biskra</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA145</td><td>A. solani s.l</td><td>Tomato</td><td>Biskra</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA146</td><td>A. solani s.l</td><td>Potato</td><td>Tipaza</td><td>-</td><td>+</td><td>S</td><td>-</td><td>-</td></tr><tr><td>DA147</td><td><strong>A. linariae</strong></td><td>Tomato</td><td>Tipaza</td><td>+</td><td>-</td><td>ND</td><td>MH243781</td><td>-</td></tr><tr><td>DA148</td><td>A. linariae</td><td>Tomato</td><td>Mostaganem</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA149</td><td>A. linariae</td><td>Tomato</td><td>Mostaganem</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr></tbody></table>

(Continued)
<table><caption>**Table 1.** (Continued).</caption><thead><tr><th>Isolate</th><th>Species<sup>a</sup></th><th>Host</th><th>Geographic origin</th><th>PCR OATF4/ OATR2</th><th>PCR OAsF7/OAsR6</th><th>PCR RFLP Profile<sup>b</sup></th><th>CAL</th><th>Rpb2</th></tr></thead><tbody><tr><td>DA150</td><td><i>A. linariae</i></td><td>Tomato</td><td>Mostaganem</td><td>+</td><td>-</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>DA152</td><td><i>A. grandis</i></td><td>Tomato</td><td>Alger</td><td>-</td><td>+</td><td>G</td><td>MH243782</td><td>-</td></tr><tr><td>DA153</td><td><i>A. linariae</i></td><td>Tomato</td><td>Alger</td><td>+</td><td>-</td><td>ND</td><td>MH243783</td><td>-</td></tr><tr><td>DA154</td><td><i>A. grandis</i></td><td>Tomato</td><td>Alger</td><td>-</td><td>+</td><td>?</td><td>MH243787</td><td>-</td></tr><tr><td>DA155</td><td><i>A. grandis</i></td><td>Tomato</td><td>Alger</td><td>-</td><td>+</td><td>G</td><td>MH243784</td><td>-</td></tr><tr><td>DA156</td><td><i>A. grandis</i></td><td>Tomato</td><td>Alger</td><td>-</td><td>+</td><td>G</td><td>MH243785</td><td>-</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Phylogenetic tree reconstructed by the maximum likelihood method from the alignment of calmodulin sequences of 42 isolates belonging to the *Alternaria* section *Porri*. Bootstrap support values greater than 80 % are indicated by arrows. The calmodulin sequences of the following strains were included as references: *A. linariae* CBS 109156 (GenBank number JQ646257), *A. protenta* CBS 116696 (GenBank number JQ646236), *A. solani* CBS 109157 (GenBank number KJ397981), *A. grandis* CBS 109158 (GenBank number JQ646249). The calmodulin sequence from *A. alternata* EGS 34016 (GenBank number JQ646208) was used to root the tree.</figcaption></figure>

### Distribution and prevalence of the large-spored *Alternaria* species in the bioclimatic regions of Algeria

The surveys carried out through the different Algerian potato and tomato growing regions revealed the

<caption>**Figure 4.** Phylogenetic tree reconstructed by the maximum likelihood method from the alignment of RPB2 sequences of 17 isolates belonging to cluster B2 in the calmodulin phylogeny. Bootstrap support values greater than 80 % are indicated by arrows. The RPB2 sequences of the following strains were included as references: *A. linariae* CBS 105.41 (GenBank number KJ718353), *A. solani* CBS 106.21 (GenBank number KJ718410), *A. protenta* CBS 347.79 (GenBank number KJ718392). The RPB2 sequence from *A. alternata* EGS 34016 (GenBank number JQ646490) was used to root the tree.</caption>

presence of *A. grandis*, *A. linariae* and the species complex *A. solani*-A. protenta at variable levels. The frequencies of these species varied according to the host plants (potato or tomato) and to the geographical locations (Table 2). *Alternaria solani sensu lato* (i.e. including *A. protenta*) was the most common species in Algeria, representing 65% of the large-spored isolates, followed by *A. linariae* (25%) and *A. grandis* with low isolation frequency (10%). Isolation frequencies of these species varied with the sampled crop, although they were all found on both potato and tomato. *Alternaria solani sensu lato* (80%) and *A. grandis* (64%) were more prevalent on potato than on tomato. Conversely, *A. linariae*

<footnote><sup>a</sup> Species names in bold characters were supported by sequence data; *A. solani* s.l stands for *A. solani sensu lato* (i.e. including *A. protenta*). <sup>b</sup> PCR-RFLP profiles: S = typical of *S. solani*; G = typical of *A. grandis*; ND = not determined; ? = atypical restriction profile</footnote>
<table><caption>**Table 2.** Frequencies of the four large-spored *Alternaria* species according to the host plants (potato and tomato) and to the geographical locations</caption><thead><tr><th>Origin</th><th><i>A. solani</i><br><i>A protenta</i></th><th><i>A. grandis</i></th><th><i>A. linariae</i></th></tr></thead><tbody><tr><td><strong>Host plant origin</strong></td><td></td><td></td><td></td></tr><tr><td>Potato</td><td>80.2<sup>a</sup>–82.8<sup>b</sup></td><td>64.3–9.6</td><td>18.9–7.5</td></tr><tr><td>Tomato</td><td>19.8–35.2</td><td>35.7–9.2</td><td>81.1–55.6</td></tr><tr><td><strong>Geographic origin (bioclimatic area)</strong></td><td></td><td></td><td></td></tr><tr><td>Mediterranean</td><td>16.7–39.0</td><td>50.0–17.0</td><td>48.6–44.0</td></tr><tr><td>Semi-arid</td><td>34.3–70.2</td><td>50.0–14.9</td><td>18.9–14.9</td></tr><tr><td>Arid</td><td>49.0–79.7</td><td>0–0</td><td>32.4–20.3</td></tr></tbody></table>

<sup>a</sup> Percentage of the total number of isolates of the same species according to the host plant or the geographic origin

<sup>b</sup> Percentage of the total number of isolates from the same host or from the same geographic origin

was more frequently isolated from tomato than from potato. Locations of sample collection could be divided into three temperature and precipitation zones (Mediterranean, semi-arid or arid). In Mediterranean and arid zones, isolates were almost equally distributed between potato and tomato. *Alternaria linariae* was over-represented in the Mediterranean zone (44% of the collected isolates) compared to the arid zone where it represented only 20% of the collected isolates. The reverse situation was observed for *A. solani sensu lato*. *Alternaria grandis* was not isolated from samples collected from the arid zone, although it represented 15% of isolates from the semi-arid zone and 17% of those from the Mediterranean zone. Only small-spored *Alternaria* isolates were obtained from samples in two locations, i.e. Ain Defla and Laghouat.

*Pathogenicity and parasitic specialization of *Alternaria* species on potato and tomato*

Fourteen isolates representing the four large-spored *Alternaria* species were used in the pathogenicity tests. These were: *A. solani* (isolates DA008, DA114, DA140 and DA141), *A. grandis* (isolates DA009, DA060, DA099 and DA152), *A. protenta* (DA062, DA082 and DA086) and *A. linariae* (DA002, DA007 and DA153). The symptoms (necroses surrounded by yellow halos) obtained on detached leaflets in Petri dish assays were confirmed by high degrees of aggressiveness obtained on whole seedling plants of potato and tomato (Figure 5). All the tested isolates produced symptoms on both plant species irrespective of their host plant of origin (Figure 6). The

<figure><img src="image_7.png" /><figcaption>**Figure 5.** Symptoms on detached leaflets (a, d) and whole tomato (c, e) or potato (b, f) plants inoculated by *A. linariae* isolate DA002 (a, b and c) or by *A. grandis* isolate DA009 (d, e and f)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 6.** Disease severity evaluated using a visual rating scale (0-100%) at 15 days post-inoculation of tomato (black bars) and potato (grey bars) leaves with isolates representing the four large-spored *Alternaria* species. Letters (P or T) under the isolates identify the host of origin (Potato or Tomato).</figcaption></figure>

degrees of aggressiveness for each host/pathogen combination varied for the isolates, however. On average, *A. solani* isolates were more aggressive on both tomato and potato, while *A. protenta* isolates were less aggressive with a marked preference for their original host plants.

DISCUSSION

Early blight has long been attributed to large-spored *Alternaria* species, i.e. *A. solani* and *A. grandis* on potato and *A. solani* and *A. linariae* on tomato. Small-spored species related to *A. alternata* are considered to be responsible for the brown spot disease (Nolte, 2008, Tymon *et al.*, 2016). The surveys carried out in Alge-
ria between 2012 and 2015 have shown the presence of
several *Alternaria* species producing large conidia in
samples from both hosts with typical early blight symp-
toms, as well as in samples with symptoms that resem-
ble brown spot. On the other hand, many Algerian iso-
lates of the section *Alternaria* collected by Bessadat *et al.*
(2017) have caused typical early blight symptoms after
artificial inoculations on tomato, and were therefore
considered pathogenic but with low aggressiveness. This
complicated the attribution of a given symptom to a giv-
en *Alternaria* species. Moreover, morphological similari-
ties between species within the *Alternaria* section *Porri*
precludes reliable identification at the species level based
only on these criteria. In the present work, molecular
tools were used to confirm the identity of large-spored
isolates originating from tomato and potato leaves. The
diagnostic species-specific primers developed by Ganni-
bal *et al.* (2014) were successfully used to quickly char-
acterized 147 isolates from a collection the 155 large-
spored isolates, and to distinguish *A. linariae* from iso-
lates that could correspond to either *A. solani*, *A. grandis*
or *A. protenta*. No amplification signal was obtained for
eight isolates either due to poor quality of the extracted
DNA or because these isolates corresponded to another
species within section *Porri*. Although never previ-
ously observed in Algeria, it has recently been reported
that *A. macrospora* may be responsible for leaf spot of
tomato in China (Zhang *et al.*, 2017). To discriminate
between *A. grandis*, on one hand, and *A. solani* or *A.
protenta*, on the other, we took advantage of sequence
polymorphism within the calmodulin gene to develop
a PCR-RFLP assay. Reliability of these diagnostic meth-
ods was checked by sequencing the calmodulin locus,
and taken together they allowed us to accurately type
more than 90% of the large-spored isolates without the
need for nucleotide sequencing. Applying these tools to
our isolates, we observed that *A. solani* and *A. linariae*
predominate on their respective potato or tomato hosts
in most of the surveyed areas. In agreement with recent
observations (Bessadat *et al.*, 2017, Ayad *et al.*, 2018), the
presence of *A. linariae* and *A. grandis* in Algeria, tradi-
tionally considered to be restricted, respectively, to either
tomato or potato, was also confirmed on these two plant
hosts. *Alternaria linariae* was found on potato and *A.
grandis* on tomato in the coastal regions of Algiers and
Tipaza, where these two crops often exist side by side or
follow each other in the same plot during the seasons.
*Alternaria grandis* has not been isolated from the Saha-
ran regions that are characterized by warm and arid
periods during each year. Analyses of nucleotidic poly-
morphism at the RPB2 locus has shown that *A. protenta*
was recorded for the first time on tomato in the coastal

region of Mostaganem (northwest of Algeria), where
the climate is very different from that of the South. This
species has already been reported on potato in Belgium
(Landschoot et al., 2017), but also in a particular region
of the south of Algeria (El Oued), where the tempera-
tures are high and the potato crops are grown under
overhead irrigation (Ayad et al., 2017).

Cross inoculations carried out with isolates repre-
senting the four identified large-spored *Alternaria*
species (*A. solani*, *A. grandis*, *A. linariae* and *A. pro-
tenta*) on potato and tomato, showed that aggressive-
ness toward the two host plants varied according to the
isolates but not with respect to particular fungal species.
Similar experiments carried out on detached leaves with
*A. linariae* and *A. grandis* (Rodrigues *et al.*, 2010; Car-
doso, 2014; Gannibal *et al.*, 2014) have previously shown
that these two species were able to cause disease on both
plants with more severe symptoms on their respective
traditional hosts. This suggests that the parasitic special-
ization of large-spored *Alternaria* species on solanaceous
crops should be reconsidered.

ACKNOWLEDGEMENTS

The authors thank the UMR 1345 IRHS, Université d'Angers-INRA-Agrocampus Ouest (France) for hosting D. Ayad, and the Ministry of Higher Education and Scientific Research of Algeria for awarding her scholarship.

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