Teratoramularia rumicis Kushwaha, Sanjeet, Sanjay & Raghv. Singh, 2021

Verma, Sanjeet Kumar, Kushwaha, Prakash, Yadav, Sanjay & Singh, Raghvendra, 2021, Morphology and phylogeny of Teratoramularia rumicis-a new foliar pathogen of Rumex crispus from India and diversity of Ramularioid complex on Rumex spp., Phytotaxa 523 (3), pp. 208-228 : 212-222

Teratoramularia rumicis Kushwaha, Sanjeet, Sanjay & Raghv. Singh , sp. nov. ( Figs. 1–5 )

MycoBank number: MB 832277

Diagnosis:— Teratoramularia rumicis differs from previously reported Teratoramularia spp. due to having longer and septate conidiophores; more septate conidia, ramoconidia with more apical hila and absence of type II conidia.

Etymology:— Latin, rumicis derived from the name of host genus.

Type:— India, Madhya Pradesh, Badamalehra South Forest, Chhatarpur , 24° 41.523’ N and 79° 20.127’ E, on living leaves of Rumex crispus L. ( Polygonaceae ), March 2016, coll. Prakash Kushwaha, AMH 9827 (holotype) , MH-DHSGU 225 (isotype). NFCCI 5008 (ex-type living culture).

Leaf spots amphigenous, initially circular to subcircular but later on irregular and spread on the entire leaf surface, silvery white and appear like powdery mildew. Colonies amphiphyllous, effuse, white crystal-like structure, whitish. Mycelium internal, hyphae branched, septate, hyaline, smooth, thin-walled. Stromata well developed, compact, erumpent, pseudoparenchymatous, hyaline, 16−34 × 16−33 µm. Conidiophores arising in fascicles from stromata, erect to slightly curved, unbranched, smooth, hyaline, 0−2-septate, thin-walled, 4−21 × 1.5−2.0 µm. Conidiogenous cells integrated, terminal, polyblastic, cylindrical, with slightly thickened and darkened loci, 0.7−1.0 µm diam. Conidia mostly catenate, forming ramoconidia, intercalary and terminal conidia, 0−2-septate, smooth, hyaline, thin-walled, hilum slightly thickened and darkened, 0.7−2.2 µm diam; ramoconidia subcylindrical to fusiform, 7–28 × 1–2 μm, with 2−5 apical hila; intercalary conidia subcylindrical to fusiform, sometimes curved, 13−25 × 1.5−2.5 µm, in chains of up to 7 conidia; terminal conidia obovoid to cylindrical, 4–10 × 2–2.5 μm.

Culture characteristics

Very slow growing, after one month of inoculation on PDA reached about 20 mm diam. at 25±5° C, top colour whitish and reverse dark blackish brown, subcircular, surface raised, folded, smooth and margin crenate. Hyphae branched, septate, thin-walled and smooth, 1.5‒2 μm diam. Conidiophores develop as a lateral branch of hyphae, sometime reduced to conidiogenous cells, subhyaline to very light olivaceous brown, thin-walled, 0‒2-septate, smooth, 10‒ 22 × 1.5‒2 μm diam. Conidiogenous cells integrated, terminal, cylindrical, thin-walled, smooth, with up to 1.2 μm thickened and darkened loci. Conidia mostly catenate, forming ramoconidia, intercalary and terminal conidia, 0−2- septate, smooth, subhyaline to very light olivaceous brown, thin-walled, hilum slightly thickened and darkened, 0.7‒ 1.5 μm diam.; ramoconidia subcylindrical to fusiform, 14‒26 × 1.2‒2 μm, with 2−5 apical hila; intercalary conidia subcylindrical to fusiform, sometimes curved, 10‒22 × 1.2‒2 μm, in chains; terminal conidia cylindrical to clavate, 7‒46 × 1‒1.5 μm.

Pathogenicity of the isolates

After 10 days of inoculation, leaf spots similar to that previously described in this manuscript were observed, whereas control plants remained healthy. Koch’s postulates were confirmed by the re-isolation of the same pathogen from inoculated healthy leaves after 10 days of inoculation. The morphological feature of reisolated pathogen was similar to that of inoculated one.

Phylogeny

Based on a megablast search of NCBI GenBank nucleotide database, the closest hits using ITS and LSU sequences were Teratoramularia rumicicola strain CPC 14653 (GenBank: KX287550 , Identities = 814/815 (99%), gap = 1/815(0%)) and Teratoramularia rumicicola strain CPC 14654 (GenBank: KX287256 , Identities = 697/707 (99%), gap = 3/707 (0%)) respectively.

TABLE 2. Comparative account of Teratoramularia spp.

Teratoramularia spp. infinita persicariae rumicicola rumicis kirschneriana

Conidiophores (µm) Reduced to conidiogenous Reduced to conidiogenous Reduced to conidiogenous 0−2-septate, Reduced to conidiogenous cells, cells, cells, 4−21 × 1.5−2.0 cells,

6.5–19 × 1–2 4.5–15 × 1–2 5–16.5 × 1–2 13.5–16 × 1–1.5

Septation Aseptate Aseptate 0−1-septate 0−2-septate Aseptate

Ramoconidia (µm) 4.5–17 × 1–2, 9.5–30 × 1.5–2.5, 8.5–23 × 1.5–2.5, 7–28 × 1–2, 7–15 × 1–2,

with 2 apical hila with 2 apical hila with 2 apical hila with 2−5 apical hila with 2 apical hila

Intercalary conidia (µm) 5–25.5 × 1–2, 8.5–30 × 1–2.5, 6.5–20 × 1.5–2.5, 13−25 × 1.5−2.5, 6–17 × 1–2,

in chains of up to 11 conidia in chains of up to 8 conidia in chains of up to 5 conidia in chains of up to 7 conidia in chains of up to 5 conidia

I

Type

Conidia Terminal conidia (µm) 3–6 × 1–2 3–10 × 1.5–3 3–8 × 1.5–3 4–10 × 2–2.5 3.5–7 × 1–2

Type II Not observed Not observed Brown Not observed Not observed

punctata ( Cucurbitaceae ) ( Polygonaceae ) Host Conyza canadensis Persicaria nepalensis , Rumex crispus Rumex crispus Setaria palmifolia (Poaceae) ( Asteraceae ), Thladiantha Fagopyrum esculentum (Polygonaceae) ( Polygonaceae )

Distribution Brazil, Taiwan South Korea South Korea India Taiwan

TABLE 3. Species of Ramularia reported on Rumex spp. (Polygonaceae)

Ramularia spp. bulgarica decipiens occidentalis pratensis pseudodeci-piens pseudorubella rubella rumicis

Superficial + – – – – – – –

secondary hyphae

Conidiophores S to F, F, F, S to F, F, F, F, F,

(µm) 8–50 × 2–4 12.5–40 × 2.5–4 5–60 × 1–3.5 (6–)10–60(–80) × (5–)10–30(–35) × 10–25 × 2–5 40–150 × 2–6 (10–)15–80(–130) 2–5 2–5 × 2–6

Conidiogenous loci St Th St Th St St St Th

Catenation C C C C C NC NC NC to C

cylindrical fusiform fusiform ovoid, oblong- at both ends ovoid, cylindrical- ovoid, subcylindric- cylindrical(-fusoid) subcylindric Shape Narrowly ellipsoid- Cylindrical, tapering Narrowly ellipsoid- Narrowly ellipsoid- Oblong, Ellipsoid, obovoid, Obovoid Subcylindric

Size (µm) 6–45 × (1.5–)2–3.5(– 16–38 × 4–7 5–35 × 1–3 (6–)8–25(–35) × (10–)20–45(–55) 12–25 × 2–6 (10–)15–35 × 6–12 (10–)12–40(–50) × 4) (1.5–)2–4(–5) × 2–5 (3–)5–8(–10)

Septation 0–2(–4) 0–3 0–1(–2) 0–3 (0–)1–3 Aseptate Aseptate, rarely 0–3

Conidia single septate

Hila St Th St Th St St St Th

Remark: + = Present, – = Absent, C = Catenate, F = Fasciculate, NC = Noncatenate, S = Solitary, St = Slightly or somewhat thickened and darkened, Th = Thickened or thickened and darkened.

The phylogenetic analysis based on ITS and LSU sequences implied that all the species of Teratoramularia were well-resolved and form five major clades in Teratoramularia viz., T. infinita Videira and Crous , T. kirschneriana Videira and Crous , T. persicariae Videira et al. , T. rumicicola Videira et al. and T. rumicis sp. nov. ( Figs. 6 and 7 ). Our proposed new species, T. rumicis , was distinguished from other species by both ITS and LSU and clustered as sister lineage to the T. rumicicola clade.

Figures

FIGURE 1. Symptoms of Teratoramularia rumicis on Rumex crispus. a. Plant showing symptoms. b. Initial stage of symptom on upper surface of leaf. c. Late stage of symptom on upper surface of leaf. d. Initial stage of symptom on lower surface of leaf. e. Late stage of symptom on lower surface of leaf. f. Symptoms of Teratoramularia rumicis after 10 days on inoculation. Scale bars b–f =10 mm.

FIGURE 2. Microphotographs of Teratoramularia rumicis (AMH9827, holotype). a–c. Fascicles of conidiophores with stromata. d. Conidia in catenation. e–j. Ramoconidia with apical hila. k–n. Intercalary conidia. o–q. Terminal conidia. Scale bars a–n = 10 µm, o–q = 5 µm.

FIGURE 3. Scanning Electron Microphotographs of Teratoramularia rumicis (AMH9827, holotype). a. Surface of leaf covered with fascicles of conidiophores and conidia.b. Single fascicle of conidiophores with conidia. c, d. Conidia with simple and branched catenations. Scale bars a = 100 µm, b = 40 µm, c = 10 µm, d = 5 µm.

FIGURE 4. Scanning Electron Microphotographs of Teratoramularia rumicis (AMH9827, holotype). a–c. Ramoconidia with 2–5 apical hila (green arrows for ramoconidia). d. Intercalary conidia in chain (red arrows for catenation). e. Terminal conidia. f. Locus of conidiogenous cell. g. Hila of conidia. Scale bars a = 10 µm, b, c = 5 µm, d = 10 µm, e = 2 µm, f = 0.5 µm, g = 3 µm.

FIGURE 5. Ex-type culture of Teratoramularia rumicis (NFCCI 5008) on PDA after one month at 25±5°C. a, b. Germinating conidia on PDA. c. Top view of culture plate. d. Reverse view of culture plate. e, f. Single colony on PDA after one month. g. Mycelium with conidiophores. h, i. Conidia in catenation. j–n. Ramoconidia with apical hila. o–r. Intercalary conidia. s, t. Terminal conidia. Scale bars a, b = 10 µm, c–f 10 mm, g–o = 10 µm, p, q = 5 µm, r = 10 µm, s = 5 µm, t = 10 µm.

FIGURE 6. Phylogram generated from Maximum likelihood analysis based on ITS region and LSU gene of strains belong to Teratoramularia. Bootstrap values for ML above 50% and Bayesian posterior probabilities support above 0.50 are indicated. Red names indicate Teratoramularia rumicis.

FIGURE 7. Phylogram generated from Maximum likelihood analysis based on combined data of genes of ITS and LSU of Teratosphaeriaceaean taxa. ML bootstrap support values above 50% and BI posterior probabilities support above 0.50 from BI are shown above the branches. The newly sequenced collection is in red.