First report of Diaporthe convolvuli on field bindweed (Convolvulus arvensis) in Russia

The genus Diaporthe Nitschke (Sordariomycetes, Diaporthomycetidae, Diaporthales, Diaporthaceae) was described in 1867^[1] with the type species D. eres Nitschke. The genus Phomopsis (Sacc.) Bubák was described in 1905^[2] with the type species P. lactucae (Sacc.) Bubák. Diaporthe species have a complex pleomorphic life cycle. Previously, Phomopsis species were considered to be the asexual stages of Diaporthe species. According to the International Code of Nomenclature for algae, fungi, and plants and the principle of "one fungus–one name", Diaporthe, being the older generic name, has priority over Phomopsis^[3]. Diaporthe encompasses important plant pathogenic, endophytic, and saprobic species, with a diverse host range and a global distribution^[4,5].

Species identification in the genus Diaporthe has evolved from host association and morphology to the widespread adoption of DNA sequencing^[4−10]. The most taxonomically informative DNA loci more frequently used to reconstruction the molecular phylogeny of Diaporthe species are the internal transcribed spacer (ITS) of the ribosomal DNA and partial calmodulin (cal), histone 3 (his3), translation elongation factor 1α (tef1), and β-tubulin (tub2) genes^[4,5,7,9,10]. A polyphasic approach for reliable species recognition should be used that relies on the consolidated species concept (CSC)^[11], which involves the incorporation of morphological, biological, and phylogenetic characteristics^[9,12,13]. Data on phylogenetic features should be obtained according to the genealogical concordance phylogenetic species recognition (GCPSR)^[14] method using a multilocus phylogenetic approach for recognizing fungal species. Certain Diaporthe species are known to produce biologically active compounds with herbicidal activity^[15−17]. For example, Diaporthe convolvuli (Ormeno-Nuñez, Reeleder & A.K. Watson) R.R. Gomes, Glienke & Crous, which is host-specific to Convolvulus arvensis L., produces secondary metabolites, namely convolvulanic acid A, convolvulanic acid B, convolvulol, and α-pyrone convolvulopyrone, which possess phytotoxic activity^[15]. Fungi with this potential could be explored given their potential application in agriculture as promising candidates for the development of natural herbicides. Field bindweed Convolvulus arvensis is a harmful weed of major agricultural crops that is distributed almost worldwide^[18]. In Russia, it is found in the European part, the Caucasus, western and eastern Siberia, and the Far East.

In July 2024, visually healthy leaves of C. arvensis were collected in northwest Russia (Pushkin, Saint Petersburg, Leningrad Region). Two Diaporthe sp. strains were isolated from these leaves. The aim of this study was to identify these strains according to the CSC by their phylogenetic, micromorphological, and cultural features and to assess their pathogenicity.

Leaves of C. arvensis were surface-sterilized by consistent rinsing with a 0.1 % AgNO₃ and sterile water and incubated on potato sucrose agar (PSA)^[19]. The Petri dishes were incubated at 24 °C in the dark and were analyzed after 7–10 days of cultivation. The obtained strains MF-SOR 61.20.1 and MF-SOR 61.20.2 were stored in microtubes on PSA at 4 °C in the mycological collection of pure cultures at the A.A. Jaczewskii Laboratory of Mycology and Phytopathology, All-Russian Institute of Plant Protection (MF, VIZR, Saint Petersburg, Russia).

DNA isolation, polymerase chain reaction, and sequencing

Genomic DNA was extracted from mycelia obtained from the cultures incubated on PSA according to the standard cetyltrimethylammonium bromide (CTAB)/chloroform protocol^[20].

The ITS region of the rDNA and partial cal, his3, tef1, and tub2 genes were amplified and sequenced for the strains studied. The primers ITS1 and ITS4^[21], CAL-228F and CAL-737R^[22], CYLH3F^[23] and H3-1b^[24], EF1-728F and EF1-986R^[22], and βtub2Fw and βtub4Rd^[25] or T1 and Bt2b^[26], were used to amplify the ITS region and the partial cal, his3, tef1, and tub2 genes, respectively. The polymerase chain reaction (PCR) conditions were as follows: 95 °С for 5 min; followed by 35 cycles of 92 °С for 50 s; 55 °C for 40 s, (ITS1/ITS4), 52 °C for 40 s (βtub2Fw/βtub4Rd and T1/Bt2b), 56 °C for 40 s (CAL-228F/CAL-737R), or 58 °C for 40 s (CYLH3F/H31-b and EF1-728F/EF1-986R); 72 °С for 75 s; and a final elongation for 5 min at 72 °С.

Single-strand DNA of amplicons were sequenced by Sanger's method on an ABI Prism 3500 analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) with a Bigdye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer's instructions. The nucleotide sequences of the ITS, cal, his3, tef1, and tub2 genes were deposited in the GenBank database with the corresponding accession numbers (Table1).

Phylogenetic analysis

Sequences were assembled using Vector NTI advance v. 11.0 (Invitrogen, Thermo Fisher Scientific) and aligned with ClustalX 1.8^[27]. The alignments were concatenated using Sequence Matrix^[28]. To implement GCPSR and estimate the possibility of combining the set of five loci, single locus trees were generated and compared to detect conflict (data not shown). A multilocus phylogenetic analysis was based on the combined ITS, cal, his3, tef1 and tub2 sequences for all strains studied and all species currently accepted in the Diaporthe section Sojae sensu Dissanayake et al.^[4] and Zhang et al.^[5], 89 species in total. Additionally, sequences for 34 species published in 2023–2025 that were omitted by these authors were included. The tree was rooted using the ex-type Diaporthella corylina Lar.N. Vassiljeva (CBS 121124). Sequences of representative Diaporthe strains and type species were obtained from GenBank (Table 1).

Phylogenetic analysis of the combined aligned data consisted of maximum likelihood (ML) and Bayesian inference. ML analysis was implemented in the IQ-TREE^[29]. Modeltest in IQ-TREE determined the most suitable nucleotide substitution model for the nucleotide dataset according to the Bayesian information criterion (BIC) model TIM2e+F+R5. Bootstrap values with 100,000 replicates were calculated for tree branches. Bayesian inference was performed by MrBayes v. 3.2.1. in ARMADILLO v. 1.1^[30] 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 10⁷ generations and sampled every 1,000 generations. The first 25% of the generations were discarded as burn-in, and Bayesian posterior probabilities (BPP) were calculated from the remaining trees.

Morphology

Pure cultures of the studied strains were incubated on PSA, oatmeal agar (OA)^[31], and Czapek agar (CZ)^[19]. Petri dishes (90 mm in diameter) were placed for 10 days under 12 h near-ultraviolet light/12 h dark to stimulate sporulation. Colony morphology and diameter were examined after 10 days. Observations and measurements of 100 conidia for each strain were conducted with an Olympus SZX16 stereomicroscope (Olympus, Tokyo, Japan) and an Olympus BX53 microscope. Images were captured with a PROKYON camera (Jenoptik, Jena, Germany) with Nomarski differential interference contrast.

Pathogenicity test

For the pathogenicity tests, leaf segments of C. arvensis were inoculated with all the studied strains. The mycelial suspension was used as the inoculum. For inoculation, strains were grown in a liquid soybean (Glycine max) nutrient medium with the following composition: KH₂PO₄ (0.2%), (NH₄)₂SO₄ (0.1%), MgSO₄ (0.1%), glucose (2%), and soy flour (1%). In total, 50 mL of the medium in 250-mL flasks was inoculated with three 5-mm mycelial discs cut from 2-week-old colonies grown on PSA and kept on an orbital shaker (200 rpm) for four days at 24 °C. The mycelium was separated from the liquid culture by filtering and then dried with filter paper and ground in sterile water to prepare a mycelial suspension of 100 mg/mL. Visually healthy C. arvensis leaves were collected from their natural habitat and cut into segments (1.5 cm × 1.5 cm) then placed in Petri dishes. Six inoculated leaf segments (replicates) were used per strain. To create a moisture chamber effect, filter paper premoistened with sterile water was added to each Petri dish. A drop of the mycelial suspension (10 µL) was applied to the center of leaves which had previously been wounded with a needle (diameter of damage: 0.5 mm) or intact leaves on the abaxial surface of the leaf. In the control, the wounded and nonwounded leaf segments were treated with 10 µL of sterile distilled water. After inoculation, closed Petri dishes with the leaf segments were kept at room temperature and evaluated for the presence of symptoms at 3, 5, and 7 d post treatment (dpt). Subsequent reisolation of the fungus from the leaf segments and identification were carried out to fulfill the Koch postulates.

A multilocus phylogenetic analysis based on the ITS, cal, his3, tef1, and tub2 sequences inferred intraspecific relationships within all species (123) currently accepted in the Diaporthe section Sojae for which nucleotide sequences are available. The total analysis included 129 strains, 2 of which were our strains and 127 were references. The total combined matrix consisted of 2,900 characters with gaps (520 bp, ITS region; 560 bp, cal; 498 bp, his3; 452 bp, tef1; and 870 bp, tub2), among which 616 (21%) characters were conserved, 1,480 (51%) were informative, and 804 (28%) were uninformative.

The analysis of the single-gene phylogenies revealed no conflicts. The individual gene trees, as well as the combined tree (Fig. 1), showed that the studied strains MF-SOR 61.20.1 and MF-SOR 61.20.2 with the ex-type CBS 124654 and representative Florida Atlantic University (FAU) 649 strains clustered in distinct monophyletic clade corresponding to D. convolvuli species.

Figure 1. 

Phylogenetic tree of Diaporthe section Sojae inferred from a ML analysis based on a concatenated alignment of the ITS region, and partial cal, his3, tef1, and tub2 genes. The ML bootstrap support values (MLBS ≥ 70%) and Bayesian posterior probabilities (BPP ≥ 0.70) are given at the nodes (MLBS/BPP). The studied strains are given in blue. The ex-type strains are marked with T. Purple lines indicate the boundaries of species complexes.

Morphology

On all tested nutrient media, the colonies of the strains reached the edge of the Petri dish within 10 days. Colonies on PSA (Fig. 2a) had sparse, silvery-white aerial mycelia generally at the colony periphery. On OA (Fig. 2b), aerial mycelia were absent. On CZ (Fig. 2c), aerial mycelia that were abundant, silvery-white, and floccose covered the entire colony, forming dense cushions. Conidiomata were pycnidial, abundant on all media, and the colonies on PSA and OA appeared mottled because of the presence of numerous pycnidia. Pycnidia were distributed evenly across the colony on all media and formed concentric rings at the periphery on PSA, and were superficial and immersed, solitary or within large conglomerates (Fig. 2d, e), often arranged in long (5–10 pycnidia on PSA and OA; Fig. 2f) or short (on CZ 3–5 pycnidia) chains. Pycnidia (Fig. 2g, h) were subglobose or globose, oval or pyriform, glabrous, ostiolate, and pappilate or with a short neck. On PSA, they were 210–386 (292 ± 14) × 140–359 (249 ± 14) μm; on OA, they were 88–364 (273 ± 17) × 95–277 (200 ± 12) μm; on CZ, they were 230–454 (343 ± 13) × 218–499 (312 ± 17) μm. The pycnidial wall was pseudoparenchymatous, composed of isodiametric cells, with the outer layers pigmented. Conidiophores reduced to phialidic conidiogenous cells (Fig. 2i, j) formed from the inner cells of the pycnidial wall, and were hyaline, smooth, and bottle-shaped, measuring 5.64–15.89 (9.69 ± 0.87) × 3.69–8.97 (5.65 ± 0.47) μm. Only alpha conidia were observed. Alpha conidia (Fig. 2k) were smooth, hyaline, guttulate, and ellipsoidal. On PSA, they were 8.1–16.2 (12.3 ± 0.1) × 3.2–4.7 (3.8) μm; on OA, they were 8.8–14 (11.5 ± 0.1) × 3.2–4.5 (3.8) μm; on CZ, they were 7.5–16.2 (11.8 ± 0.2) × 3.3–5 (4.1) μm. Ascomata were absent.

Figure 2. 

Diaporthe convolvuli strain MF-SOR 61.20.1 at 10 days of growth. (a)–(c) Pure cultures: left half, front; right, reverse. (a) PSA. (b) ОА. (c) CZ. (d), (e), (g), (h) Pycnidia on ОА. (f) Pycnidia on OA aggregated in chains. (i) Conidiogenous cell. (j) Conidia on a conidiogenous cell. (k) Conidia. Scale bars: (d), (e) = 1 mm; (f) = 200 μm; (g)–(h) = 100 μm; and (i)–(k) = 10 μm.

Pathogenicity

The pathogenicity of D. convolvuli was tested using C. arvensis leaf segments (Fig. 3). No distinct necrosis was observed on the intact sides of the segments. The first significant symptoms, namely necrosis, on the wounded abaxial surfaces of the leaf segments were observed at 3 dpt. At 3 dpt, inoculation with the strain MF-SOR 61.20.2 led to the development of tan to brown necrotic lesions measuring 0.6 ± 0.3 mm in diameter on leaf segments. At 5 dpt, the lesions measured 2.0 ± 0.5 mm. By 7 dpt, lesion size did not increase. In contrast, the strain MF-SOR 61.20.1 induced brown to black necrotic lesions. At 3 dpt, the lesions measured 5.2 ± 0.4 mm in diameter. A marked increase was observed by 5 dpt, with the lesions reaching 11.9 ± 1.3 mm. Subsequently, lesion expansion slowed, and at 7 dpt, the lesions measured 12.25 ± 0.81 mm (Fig. 4). To confirm Koch's postulates, fungal cultures were reisolated in all cases from the necrosis formed on the leaf segments. These isolates had morphological characteristics identical to those of all the strains tested.

Figure 3. 

Pathogenicity of Diaporthe convolvuli on leaf segments of Convolvulus arvensis at 5 dpt. The first line in each set (Petri dish) contains the inoculated intact abaxial part of the leaf segments; the second line is the abaxial part wounded with a needle. Inoculation variants: (a) Inoculation with sterile water; (b) mycelial suspension of MF-SOR 61.20.1; (c) mycelial suspension of MF-SOR 61.20.2.

Figure 4. 

Diameter of necrosis (mm) caused by Diaporthe convolvuli strains on Convolvulus arvensis leaf segments at 3, 5, and 7 dpt. Error bars represent standard errors of the means.

Two studied strains isolated from C. arvensis, MF-SOR 61.20.1 and MF-SOR 61.20.2, were definitively identified as D. convolvuli. Phylogenetically, these strains were closely related to the ex-type (CBS 124654) and representative (FAU 649) D. convolvuli strains, and their morphological features were consistent with that of D. convolvuli^[32]. Diaporthe convolvuli was originally described as Phomopsis convolvuli Ormeno-Nunez, Reeleder & A.K. Watson in 1988^[32] as the causal agent of leaf spot and anthracnose in field bindweed in Montreal (Canada). To our knowledge, this is the first finding of this fungus in Russia (Saint Petersburg, Pushkin) and the third in the world. The first report from Canada^[32] was of a strain that was later patented for the biological control of C. arvensis^[33]. The second was from Turkey^[34]. Furthermore, this study provides the first description, refinement, and comprehensive illustration of the fungus's cultural and micromorphological characteristics since 1988.

Both strains exhibited pathogenicity to field bindweed leaves under laboratory conditions. This observation suggests that they might be worth investigating further as potential candidates for biological weed control. The fact that these strains were isolated from visually healthy leaves but demonstrated pathogenicity under laboratory conditions indicates a latent infection in the original plant material.