First report of Nigrospora humicola (Apiosporaceae, Xylariales) on Phragmites plant from North Asia

Nigrospora Zimm. (Ascomycota, Sordariomycetes, Xylariales) are ubiquitous fungi revealed on plants^[1−4], lichens^[5], insects^[6,7], marine organisms^[8], humans^[9], and in the soil^[10]. These fungi are associated with different plants as endophytes, saprobes, parasites, and pathogens^{[2,4,11−13]}.

Nowadays, with the emerging possibilities of accurate species identification of fungi, studying the patterns of their occurrence and abundance, as well as various characteristics is an urgent task. In recent years the revision of Nigrospora genus was carried out and new species were described on the basis of the molecular and morphological evidence^{[1,4,10,13−17]}. The identified genetic diversity of Nigrospora indicates that many species of this genus have not yet been discovered. Moreover, the information on the distribution and host plants of known Nigrospora species remains often fragmentary. At least 30 Nigrospora species were detected in China on various substrates^{[10,11,13,14,16,17]}, among them, 14 Nigrospora species were associated with Poaceae plants^[11,13,14].

In Russia, the N. gorlenkoana Novobr. occurrence on wheat, barley, oats, maize, and rapeseed were previously confirmed by molecular genetic methods^[18], while there is no information on the distribution of other Nigrospora species. In our previous study, we suggested that one strain of Nigrospora sp. MFG 70052, isolated from Phragmites australis stem can be described as a potential new species based on morphology and phylogenetic analysis data, which represented the distinct genetic line, sister to N. chinensis Mei Wang & L. Cai^[18].

The aim of study was to identify the Nigrospora sp. strain isolated from Phragmites australis collected in North Asia.

The Nigrospora sp. strain was isolated from the Phragmites australis collected in Primorsky Krai of Russia in 2010. The fragments of stems with necrosis were carefully washed with tap water, and were surface sterilized by soaking in 5% NaOCl (3 min), and then rinsing with sterile water. Then, the plant tissues were placed on potato sucrose agar (PSA) containing 1 mL/L mixture of antibiotics (HyClone, Austria), and 0.4 μL/L Triton X-100 (Panreac, Spain). Following incubation at 25 °C, the pure Nigrospora culture was sub-cultures onto fresh PSA. Suspension 20 μL consisting of 20−50 conidia and sterilized distilled water was transferred to a Petri dish on PSA containing Triton X-100 and spread with a sterile spatula over the surface of the medium prepared the day before. Incubation continued for 48 h at 25 °C, after one germinated spore was selected and transferred to a new dish with PSA. The fungal growth was recorded after 3 d of incubation. The single-spored Nigrospora strain MFG 70052 was deposited in the culture collection of the All-Russian Institute of Plant Protection (VIZR, St. Petersburg, Russia).

Molecular genetic analysis

Genomic DNA was extracted from fungal mycelium grown on PSA (7 d, 25 °C, in the dark) using the CTAB protocol^[19]. The fragments of the internal transcribed spacer (ITS), β-tubulin gene (tub), and translation elongation factor EF-1a (tef) were amplified with ITS1/ITS4^[20], T1/T2^[19], and EF1-728F/EF1-986R^[21] primer pairs and according to the authors' protocols, respectively. The sequencing of the fragments was carried out on an ABI Prism 3500 sequencer (Applied Biosystems, Japan) using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, USA).

The obtained combined sequences of three loci (ITS, tub, and tef) as well as the sequences of representative 88 Nigrospora spp. strains (Table 1) were aligned using the MEGA X 10.1 program. The bestfitting substitution model was determined using the IQ-TREE 2 v.2.1.3 program. The T92+G model was chosen for the multilocus analysis. The phylogenetic analysis was performed using maximum likelihood (ML) also in IQ-TREE 2 v.2.1.3 program. Bootstrap supports values were calculated with 1,000 replicates. To further infer phylogenetic relationships among taxa, a Bayesian analysis was conducted with MrBayes 3.2.1 using 2,000,000 generations of Markov chain Monte Carlo (MCMC), and trees were sampled at every 1000^th generation.

Morphological study

The morphological characteristics of Nigrospora sp. strain MFG 70052 were observed during its growth on PSA in the dark at 25 °C for 7–30 d, while the diameter of the colony was measured on the third day of cultivation in two perpendicular directions and the average diameter of strain colony was calculated. The micromorphological structures were investigated and documented using Olympus ВХ53 microscope (Olympus America, USA) connected to a SUBRA camera (Jenoptik, Germany).

Phylogenetic analysis of the adjusted and aligned sequences of Nigrospora sp. MFG 70052, as well as reference strains, included the following lengths: ITS − 459 bp, tub − 588 bp, tef − 566 bp. The number of parsimony-informative sites per genome locus was 58 (12.6%), 184 (31.3%), and 305 (53.9%), respectively. The topology of the phylogenetic tree was concordant with that reconstructed previously^[1,2,10]. The analyzed strain MFG 70052 formed a clade with the reference strains N. humicola Qin Yang & Ning Jiang CFCC 56884 and CFCC 56885 with high bootstrap support ML/BP 100/1.0, which confirms the belonging of the strain MFG 70052 to the N. humicola species (Fig. 1).

Figure 1. 

Maximum likelihood (ML) phylogenetic tree based on DNA sequence data from three loci (ITS, tub, and tef) of Nigrospora fungi. ML bootstrap support values > 70 %, followed by Bayesian posterior probability (BP) scores > 0.95 are shown at the nodes. Thickened lines indicate ML/BP of 100/1.0. The tree was rooted on sequences of Apiospora arundinis CBS 114316. The analyzed strain is indicated in bold. T, ex-type strain.

Description morphology

The colony of Nigrospora humicola MFG 70052 reaching 80 mm on the third day of cultivation on PSA at 25 °C. Mycelium is flat, floccose, edge entire, initially white, later becoming gray when sporulation is abundant, and the edge is smooth/filamentous (Fig. 2). The reverse is smoke-gray, with age a noticeable pattern of dark hyphae and sporulation is noted.

Figure 2. 

Nigrospora humicola MFG 70052 (PSA, 25 °C in the dark). Upper surface and reverse overview after growth for (a) 7 d, and (b) 30 days. (c)−(h) Conidiophores and conidiogenous cells giving rise to brown-black conidia. (i) Septate hyphae and spherical vesicles aggregating in conidiophores. (j)−(p) Sterile cells. Scale bars = 20 μm.

Hyphae 2.5–6 μm, smooth, branched, septate, hyaline, some becoming brownish with age. Setae not observed. Conidiophores are mostly reduced to conidiogenous cells, macronematous, aggregated in clusters on hyphae. Conidiogenous cells 4–10 × 4–8 μm, hyaline to pale brown, their coloration is especially noticeable when they are aggregated, ampulliform, spherical, or subspherical.

Conidia 10.8–16.7 × 11.2–18.7 μm (13.7 × 15.6 μm), solitary, acrogenous, spherical or broadly ellipsoidal, egg-shaped, aseptate, pale brown at the beginning of formation, later black, shiny, smooth, aseptate. In some light-colored young conidia of N. humicola the presence of equatorial slits is noticeable. Sterile cells 27.0–40.8 × 11.2–21.4 μm (33.6 × 16.7 μm), terminally formed on hyphae, dark brown to black, elongated-ellipsoid or club-shaped, sometimes curved or angular.

At present, investigations of fungal diversity rely almost invariably on sequencing several informative genome loci and the development of multilocus phylogeny, for this approach provides greater resolution than the sum of morphological features. In this study, using the phylogenetic analysis of the ITS, tub, and tef loci Nigrospora strain MFG 70052 isolated from reed in 2019 from the Primorsky region of Russia bordered with China, was accurately identified as N. humicola. This is the first finding of this species on the plant (Poaceae) and the first detection in Russia. Nigrospora humicola was described recently when two strains isolated from soil in 2021 in the Hebei Province of China were studied in detail^[10].

Some Nigrospora species are associated with different plants, e.g. N. oryzae (Berk. & Broome) Petch is the most ubiquitous species of this genus and has been reported from 40 different plant host genera including Poaceae family plants^[11], and largely distributed in the territory of Russia, N. gorlenkoana was found in mycobiota of cereal crops and rapeseed plants^[18]. Overall, Nigrospora species lacks host specificity^[14], so the host range of N. humicola may also be wide.

Morphologically, MFG 70052 strain similar to N. humicola strains described by Zhang et al.^[10] in the cultural features, sizes and shapes of conidiogenous, and conidial cells. However, there are some morphological differences that we consider important for identification of this species. One of them was a formation of sterile cells that looks like conidia due to dense dark pigmentation, but they are much larger and have an irregular shape. The sterile cells are formed directly on hyphae, not on conidiogenous cells and frequently observed in some Nigrospora species^[11]. These morphological structures were described in the phylogenetically closest to N. humicola species N. chinensis, representing the sister clade^[11]. In our opinion, they are an additional diagnostic feature. Moreover, in the culture of N. humicola, stained clusters of conidiophores and conidiogenous cells are formed, the same as described in N. oryzae and N. lacticolonia Mei Wang & L. Cai^[11]. Equatorial slits are present in some young conidia of N. humicola, as in N. gorlenkoana^[11], but not mentioned in the description of the species^[10].

The results obtained in this study indicate that analysis of morphology is likely to be useful in refining our understanding of the Nigrospora species limits. The accurate identification of Nigrospora species is possible only by combined analysis of genetic, morphological, ecological, and physiological data^[2,11,22]. The diversity of Nigrospora species in nature as well as their characteristics require further investigation.