Algeria • Ain Témouchent City, Chabaat El Lahame, from leaves of wild Daucus carota. 08 April, 2021, N. Bessadat, Living culture NB968.

Colonies on PCA velvety to cottony colonies, mostly mycelial, subhyaline, loosely wooly, reaching 75 mm in diameter after 7 days; meager sporulation or lacking until hyphae are disturbed or scarified. Conidiophores emerging from the surface of agar or aerial vegetative hyphae scattered or clustered on cut agar in light-exposed areas. Primary conidiophores mostly simple, rarely branched, 25–75 × 5–6 µm, geniculate with 2–3(–4) conidiogenous loci. Secondary conidiophores, short, 3–7 × 3 µm, 1–2-celled, formed apically or sometimes laterally from primary conidia, mainly with one conidiogenous locus. Further geniculate extensions and conidium production yield several clusters of sporulation at the colony center. Sporulation pattern in single clumps of a few short branching chains consisting of 5–7 conidia. A high percentage of conidia solitary at any age of growth. Conidia ellipsoid to ovoid, with a rounded base and tapered apex; multiple transverse and longitudinal septa, conspicuously constricted near 2 or 3 transverse septa, in short chains. Mature conidia with 2–3 transverse septa and 0–3 longitudinal septa located mainly at the center of the colony, 20–24 × 8–15 µm; slightly older conidia, 25–34(–48) × 11–16 (–22) µm with 3–4(–5) transverse septa and 1–3 oblique or longitudinal septa. Conidial body color brown with relatively dark and thick transverse septa (Fig. 2A, B); the outer wall smooth or punctate. Formation of protoascomata and chlamydospores in the surface of vegetative mycelium conspicuous after 2–3 weeks of incubation. Chlamydospores hyaline, spherical or ovoid, terminal, sub-terminal or intercalary, measuring 13.5–22.5 × 10–15 µm, mostly single-celled (Fig. 2C). Protoascomata subspherical to ovoid, light brown with numerous outgrowths of hyphae, measuring 40–58 µm in diameter (Fig. 2D).

Figure 2. 

Morphology of Alternaria eureka (strain: NB968). Conidiophores and conidia on PCA for 7 days at 22 °C (A) and on inoculated carrot leaves after 24 days (B); Chlamydospores (C) and protoascomata (D) on PCA for 18 days at 22 °C. Colony phenotypes on PDA (E), PCA (F), MEA (G), and OA (H) for 7 days at 25 °C. Scale bar: 25 µm (A–D); 10 mm (E–H).

Culture characteristics at 25 °C in 7d—Colonies on PDA cottony, grayish yellow to yellowish grey (3C3/3D2) with white regular margins, attaining 60.5 ± 1.1 mm diam. (Fig. 2 E); reverse olive brown to yellowish brown (4F6/5F5). On PCA, velvety to glabrous, olive brown (4E5), 67.4 ± 0.4 mm diam. (Fig. 2F); reverse olive brown (4E4). On MEA, cottony, dull green (28D3) with a greenish-grey surface (28C2) and white regular margins, 51.9 ± 0.7 mm diam. (Fig. 2G), reverse yellowish brown to brownish orange (5F5/5C4). On OA, velvety with a cottony center, yellowish brown to dark blond (5E4/5D4), 60.6 ± 1.5 mm diam. (Fig. 2H); reverse brownish grey to yellowish brown (5F2/5E4). Sporulation of NB968 isolate occurred mainly in PCA and PDA after 7 days of incubation. It was poor on MEA and OA.

The multilocus phylogeny revealed that NB968 significantly clustered with A. eureka (ex-type, CBS 193.86), both forming a branch separated from other species in section Eureka. Cultural characters, sporulation patterns, and conidia shapes of A. eureka are different from other members of section Eureka. A high percentage of conidia have conical apical cells but differ in size and number of transverse septa from A. hungarica (15–30 × 12–13 vs. 25–48 × 8–22, respectively). Conidiogenous axes, conidiophores, and conidia chains of the latter species are longer than those of A. eureka (30–352 × 3.8–5 µm vs. 25–75 × 5–6 µm, respectively). Conidia chain formation of A. eureka is rare, while in A. hungarica, short-branched chains of 3–4 units are conspicuous. Alternaria eureka and A. hungarica conidia consist of numerous longitudinal and/or oblique septa forming multi-celled segments; the two species are, however, distinct from each other according to the sporulation pattern (see A. hungarica notes). Alternaria eureka is the only species from section Eureka known to have a sexual state (Simmons 1986); it also produces hyaline chlamydospores and sclerotia upon aging, which are lacking in A. hungarica and A. cumini.

Algeria • Mostaganem City, Kheir Eddine, from leaves of cultivated Daucus carota. 01 December, 2020, N. Bessadat, Living culture NB968; ibid. Algeria, Ain Témouchent province, Bouzedjar, from leaves of wild Daucus carota. 02 February, 2020, N. Bessadat, Living culture NB803.

Colonies on PCA velvety, approximately 70 mm in diameter, with three discrete concentric rings of growth after 7 days. Aerial axes abundant and reaching a size length of 113–387 µm, with 5 to 10 lateral conidiogenous branches and tips in light-exposed zones of young parts of the colony (Fig. 3A). These branches most frequently on the upper portions of aerial conidiogenous elements, forming an arachnoid layer of branching hyphae. Primary conidiophores formed outside the center of the colony, simple, rarely branched, produced at the substrate surface, conspicuous, straight, or geniculate, up to 30–110(–352) × 3.8–5 µm, often developing through geniculate extensions. Each conidiophore bearing 1–3 conidia, sometimes with additional short chains of 2–3 conidia through secondary conidiophores formed mainly from distal terminal conidial cells (Fig. 3B). Secondary conidiophores short, 20–52(–105) × 3–5 µm, with one or two conidiogenous sites. A high percentage of conidia of any age remaining solitary. The sporulation patterns in small clumps on elongate-branched sporulating hyphae forming a brown layer at the center of the colony. Conidia medium brown to brown, short ellipsoid, ovoid, muriform, mainly beakless or with a rounded apex (Fig. 3B). Dominant size range: 22–37(–40) µm long × 13–20 μm width with 2–4(–5) transverse septa and 0–2(–3) longitudinal septa in each of the transverse segments. In the center of the colony, a high percentage of dark pigmented conidia mature of smaller size, 17.5–25 × 12–17.5 µm, initiating chain development. All maturing conidia distinctly constricted at their transverse septa, developing thick and dark-brown pigmented outer walls and sometimes with a punctate surface.

Figure 3. 

Morphology of Alternaria hungarica (strain: NB803). Sporulation patterns on PCA for 14 days at 22 °C (A); Conidiophores and conidia on PCA for 7 days at 22 °C (B); Colony phenotypes on PDA (C), PCA (D), MEA (E), and OA (F) for 7 days at 25 °C. Scale bar: 25 µm (A, B); 10 mm (C–F).

Culture characteristics at 25 °C in 7d—Colonies on PDA cottony compact, greyish green (30E5/30E7) with irregular margins, attaining 67.5 ± 0.6 mm diam. (Fig. 3C); reverse olive (3E4). On PCA, velvety, olive brown (4E4), 69.4 ± 0.5 mm diam. (Fig. 3D); reverse grayish same color. On MEA, cottony, olive (1E3) with a grayish surface, irregular margins, 65.1 ± 0.6 mm diam. (Fig. 3E); reverse olive brown (4D3). On OA, cottony, grey to dull green (29D4) with a grayish surface, 63.8 ± 1.0 mm diam. (Fig. 3F); reverse olive brown (4E3). Sporulation of studied isolates occurs in all tested media after 7 days of incubation. It was abundant on PCA and PDA but moderate on MEA and OA.

NB803 and NB898 formed a well-supported clade with A. hungarica (ex-type, CBS 123925) phylogenetically close to A. cumini (ex-type, CBS 121329). Between these three strains and A. cumini (ex-type, CBS 121329), there were 2/464 differences in ITS, 9/529 in gpd, 10/833 in rpb2, and 2/199 in tef1. Morphological features of two isolates from carrot leaves (wild and cultivated) are similar to A. hungarica and did not contradict descriptions of Toth et al. (2011). The conidial and sporulation characteristics resemble those of CBS 123925 but with some variations in cultural characteristics (colony color and texture on PDA). Isolates NB803 and NB898 form a less dense mycelium but are darker than CBS 123925. Cultural characters, sporulation pattern, and conidia shape of this species are different from A. cumini (17.5–40 × 12–20 vs. 50–90 × 13–23 μm, respectively). The latter species produce short and erect conidiophores of 18–60 × 5–7 μm (Nishikawa and Nakashima 2020), while A. hungarica form long geniculate conidiophores (30–352 × 3.8–5 µm). A high percentage of conidia are in clumps or short chains in A. hungarica, while A. cumini produces conidia in simple chains. Both species produce dark yellow (4C8) pigmentation on PDA after 7 days, which is lacking in A. eureka.

Name refers to the organ from which the species was isolated, carrot roots.

Algeria • Oran market on infected roots of Daucus carota. 16 July, 2020, N. Bessadat, (INH001054, holotype), preserved in a metabolically inactive state via deep freezing at INH herbarium, France, using the COMIC technical platform, ex-type cultures (CBS 149902, NB830).

On PCA, attaining 75 mm in diameter, velvety (NB830, NB936) to flat (NB794), sometimes with granular appearance by the presence of abundant intra-hyphal, dematiaceous, thick-walled chlamydospores after 8–14 days. Aerial mycelium sparse and submerged hyphae abundant, producing chlamydospores in culture formed from fertile hyphae with 3–5 transverse septa and sometimes one longitudinal. These fructifying elements arising from a distinct radial system of hyphae or near the substrate surface. Conidiophores arising directly from lateral and apical aerial axes, simple, septate, 20–30 × 3.5–5 μm, straight or geniculate at successive sites of conidium production, size of cells decreasing towards apex, rarely branched, cell walls thicker than those of vegetative hyphae, pale brown to brown in color, sometimes swollen at the base, rarely from chlamydospores. Conidiogenous branches, formed on fertile hyphae, 1–2-celled, 15–20 × 3 µm; the longest conidiophores reaching 31–88 μm in length. Each usually bearing only a single conidium in young cultures and occasionally a short geniculate extension with a second or third conidium. Conidiogenous sites terminal or intercalary, proliferating sympodially, brown. Occasional chain formation of two spores through secondary conidiophores on the tip or basal cells of primary conidia. Conidia mature in broad-cylindrical morphologies, usually narrow ellipsoid, ovoid, or cylindrical with rounded base and apex, as large as 18–26 × 7–10 µm, with 3(–4) thickened transverse septa and rarely one longitudinal septum, representing a high percentage of the mature population (Fig. 4A, B). Conidia occasionally forming chlamydospores during germination, exhibiting single-celled, spherical or oval brown structures on both ends of conidia, measuring 7–12 µm in diameter (Fig. 4C). Another population of conidia with 4–5(–6) transverse septa, smooth-walled, cylindrical to obclavate, mostly straight, sometimes curved with middle cells slightly enlarged, pale brown to brown, with rounded basal and apical cells, 30–36 (–38) × 7–10(–12) and 0–1(–2) longitudinal septa in 2–3 of the transverse segments. A few conidia with 7 transverse septa, usually without longitudinal septa, 35–39 × 10–11 µm, relatively narrowed cylindrical, not forming secondary conidiophores (Fig. 4D, E). Conidiophores emerging from the surface of dead infected plants rigid, brown, clustered, or scattered, with 1–4(–6) conidiogenous loci, 25–67 µm long and 3.8–6 (–7.5) μm thick (Fig. 4F). Most conidia with 8–10(–11) transverse septa, 44–55 (–59) × 8–10 µm. On aged parts of the colony, conidia usually clustered in small clumps with 2–4 spores near the tips or laterals of primary conidiophores (Fig. 4G).

Figure 4. 

Morphology of Alternaria radicicola sp. nov. from Daucus carota: Conidia on PCA for 7 days at 22 °C (A, B, D); Chlamydospores from germinating conidia (C); Conidia and conidiophores on inoculated coriander leaves at 7 DAI (E, F); Sporulation patterns on PCA for 14 days at 22 °C (G); Chlamydospores in the surface of vegetative mycelium (H); Colony phenotypes on PDA (I), PCA (J), MEA (K), and OA (L) for 7 days at 25 °C. Scale bar: 25 µm (A–H); 10 mm (I–L).

All isolates producing dark-brown, compound chlamydospores, looking like thickened oval or rounded cell chains with punctate ornamentation, arranged in chains (Fig. 4H). Such cells conglomerating and elongating to a pearl necklace or an irregular shape, as large as 15–41 × 20–53 µm with 3–8 cells; these structures enlarging as the colony ages and observed also on six-week inoculated leaf debris in contact with soil. Conidia color before full maturity dark yellow, which deepens to an olive brown, paler towards apex, against which the thickness of major transverse septa contrasts strongly.

Culture characteristics after 7 days—Colonies color and aspect of the holotype strain on PDA (Fig. 4I), PCA (Fig. 4J), MEA (Fig. 4K), and OA (Fig. 4L) are provided in Table 2. This strain grows over a wide range of temperatures with varying growth rates. Among the different temperature and culture media, the best mycelial growth occurs at 25 °C on PDA and PCA. On all media, the strain grows slowly at 4 °C and 35 °C, moderately at 16 °C, rapidly between 20–30 °C, and does not grow at 40 °C.

Algeria • Oran City, Oran province Market, from the root of Daucus carota. 18 February, 2020, N. Bessadat, (CBS 149906, preserved in a metabolically inactive state in the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands). Living culture NB794.

Algeria • Mascara City, Tizi province, from leaves of cultivated Daucus carota. 21 December, 2020, N. Bessadat, Living culture NB936.

Phylogenetic analyses indicated that Alternaria radicicola sp. nov. fell in an individual branch close to A. tellustris (ex-type, CBS 538.83) and A. chlamydosporigena (CBS 341.71). Although the three species shared identical ITS sequences, there were 8/529 differences in gpd, 8/833 in rpb2, and 8/199 in tef1 between isolates of A. radicicola sp. nov. and A. tellustris and 8/529 differences in gpd, 11/833 in rpb2, and 7/199 in tef1 between isolates of A. radicicola sp. nov. and A. chlamydosporigena. Although A. radicicola sp. nov. has almost the same conidia size and shape as the closely related A. chlamydosporigena (Table 3), it differs from this species by producing chlamydospores in culture that are able to form fertile conidiophores; association of conidiophores with the chlamydospores has never been observed on A. chlamydosporigena (Simmons 1971; Marin Felix et al. 2019). Other species from section Embellisia exhibited variable conidial size; A. embellisia produces chlamydospores in pairs or chains (up to seven cells) (Delgado Ortiz et al. 2019), while A. radicicola isolates form shorter chains of 3–4 cells after 7 days under similar incubation conditions. Alternaria tellustris produces obclavate or long ellipsoid conidia smaller than the new species (18–33 × 6–8 vs. 20–38 × 7–12, respectively); obclavate conidia were rare or mainly immature in A. radicicola sp. nov. Chlamydospore production was influenced by temperature and time of incubation. A. radicicola sp. nov. isolates exhibited abundant chlamydospores beyond 7 days when temperatures ranged between 30–35 °C and 2–3 weeks when incubated at 20–25 °C. These structures were also observed on infected host leaves after 6 weeks of inoculation.

Name refers to conidial shape and size, which is longer than other species within the section Embellisioides.

Algeria • Mostaganem, Hassi Mamache on infected leaves of Solanum lycopersicum. 22 May, 2015, N. Bessadat, (INH001055, holotype), preserved in a metabolically inactive state via deep freezing at INH herbarium, France, using the COMIC technical platform, ex-type culture (CBS149901, NB354).

On PCA, attaining 69 mm diam., colony wooly, loose, with aerial branched subhyaline hyphae and 2–3 pairs of moderately defined concentric rings of growth and sporulation. During an initial 5–7 d of growth, colony producing only minor sporulation near the agar surface. At the same time, abundant, long, suberected aerial hyphae arising throughout light-deprived parts of the colony. The tip and some branches of these slender aerial axes enlarging into well-defined conidiophores with few lateral branches, mostly near the hyphal apex, from a simple and short conidiophore bear 2–4 conidia (Fig. 5A, B), yielding to an open layer of sporulation in the surface of the colony. Sporulation pattern forming compact, small clumps of conidia. Conidiophores septate, simple or sparingly branched, straight to slightly curved, pale to medium brown, with series of 2–6(–14), geniculate, sympodial conidiogenous sites. Primary conidiophores of short length, 15–20 × 4–7 µm, cylindrical, 0–3-septate, produced from fertile hyphae, commonly becoming 35–80 (–170) µm long with 4–9 transverse septa. Each conidiophore bearing a single conidium, rarely a chain of 2 conidia in undisturbed young colonies. Conidiogenous cells terminal or intercalary, solitary or proliferating sympodially (Fig. 5B). Seldom production of secondary conidiophores from primary conidia forming short 5–23 (–41) × 3.5–7.5 µm, 0–3-septate, cylindrical or angular complexity structure at the apex leading to false conidia chains upon the aging parts of the colony. Mature conidia with 4–7 transverse septa, 31–60 × 10–18 µm, oblong, ellipsoid, or subcylindrical, broadly rounded at the base with a bluntly rounded apical cell (Fig. 5C, D). Conidia at full development tapering gradually from narrowly ovoid into narrowly cylindrical, 56–90 (–100) × 12.5–17 µm, always rounded at base tapering towards apex with 8–11 (–15) transverse septa (Fig. 5E). A few submedian cells increasing in width and producing thin longitudinal and oblique septa. Abundant juvenile conidia, 5–20 × 2.5–12.5 µm, spherical to ovoid at the center of the colony at 14d, formed through extension of secondary conidiophores and usually 1–2-celled. Fully developed conidia mostly equilateral until enlargement of a few body cells and their secondary internal septation cells introducing minor degree of curvature. Conidia slightly, to distinctly constricting at their 1–3 transverse septa, contrastingly darker than others. One to two longitudinal septa very pale inserted in 1–3 first transverse segments. Conidia color pale to medium yellow, appearing quite smooth, thin-walled due to a lack of ornamentation on the surface. Conidium germination after 24 h usually bipolar but not conspicuous from a non-polar cell. Conidiophores emerging from the surface of dead infected plants brown, scattered, with 1–3 (–6) conidiogenous loci, 25–67 (–100) µm long and 3.8–6 (–7.5) μm thick (Fig. 5F). Most conidia with 8–10 (–11) transverse septa, 44–55 (–59) × 8–10 µm.

Figure 5. 

Morphology of Alternaria longiformis sp. nov. from Daucus carota: Conidia on PCA for 7 days at 22 °C (A–E); Conidiophores and sporulation patterns on PCA for 7 days at 22 °C (B); Conidiophores on inoculated carrot leaves at 21 JPI (F); Colony phenotypes on PDA (G), PCA (H), MEA (I), and OA (J) for 7 days at 25 °C. Scale bar: 25 µm (A–F); 10 mm (G–J).

Culture characteristics after 7 days— Colonies color and aspect the holotype strain on PDA (Fig. 5G), PCA (Fig. 5H), MEA (Fig. 5I), and OA (Fig. 5J) are provided in Table 2. This strain grows over a wide range of temperatures with varying growth rates. Among the different temperature and culture media, the best mycelial growth occurs between 20–25 °C on PDA. On all media, the strain grows slowly below 4 °C, moderately at 16 °C and 30 °C, rapidly between 20–25 °C, and does not grow well at a temperature of 35 °C. Mycelial growth was inhibited at 40 °C.

Algeria • Mascara City, Tizi province, from leaves of Daucus carota. 21 December, 2020, N. Bessadat, (CBS 149905, preserved in a metabolically inactive state in the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands). Living culture NB930.

Phylogenetic analyses indicated that Alternaria longiformis sp. nov. fell in an individual branch close to A. lolii (ex-type, CBS 115266). Between this species and A. lolii, there were 4/464 differences in ITS, 3/529 in gpd, 15/833 in rpb2, and 8/199 in tef1. Isolates of Alternaria longiformis sp. nov. are morphologically similar to A. lolii (Bessadat et al. 2021). Conidia of both species are quite similar in shape but different in size (Table 3), and primary conidiophores are geniculate but slightly different in size (25–150 × 3–5 in A. lolii and 35–170 × 4–7 μm in A. longiformis). These two species can be distinguished mainly by their sporulation patterns, the presence/absence of cellular knots, and the abundance of secondary conidiophores. Alternaria lolii was reported to produce distinctive submerged knots of hyphal cells and emergent rhizoidal branches (Simmons 2004). This species produces rarely chlamydospores (Table 3). None of these structures were observed on the two A. longiformis sp. nov. isolates. Further, branching through secondary conidiophores is conspicuous in A. lolii, which is not in A. longiformis sp. nov. Other species belonging to the same section, such as A. proteae, A. novae–zelandiae, and A. hyacinthi, form multi-geniculate conidiophores (Hoog and Muller 1973; Simmons 1990) and shorter conidia compared to A. longiformis sp. nov. (Table 3). Alternaria planifunda and A. tumida were reported to produce conspicuous chlamydospores and smaller, solitary conidia (Simmons 1983), making them distinct from the new species (20–28 × 10–13 and 35–42 × 13–18 vs. 40–97 × 10–25, respectively).