New Species of Preussia from Sedimentary Cost in Basrah Province, Iraq

A novel undiscovered fungal species was obtained from brackish environments located in Al-Faw city, situated in the southern region of Basrah, Iraq, through phylogenetic assessments of the ITS and TEF1α genomic regions. Sediment sample was collected from the seacoast of Al- Faw region and cultured on Potato carrot agar (PCA) and Potato dextrose agar (PDA), then incubated at 25 °C for 14 days. It was ascertained that this species clustered within the genus Preussia. Subsequent in-depth examinations of its morphological and anatomical features corroborated its distinctiveness. This previously unknown species is introduced here as P. aseelix. One of its notable characteristics is the absence of a true fruiting body, which is replaced by an asexual state represented by pycnidia.

Introduction

Microorganisms adapted to halophilic environments flourish in the challenging conditions found in these habitats, having developed molecular and cellular mechanisms to manage factors like osmotic pressure and reduced water activity. Such environments are teeming with fungal species that remain unidentified, demonstrating a promising area for further exploration (Sayedet al., 2020; Wingfieldet al., 2023).

Ascomycota is one of the largest fungal phyla and encompasses approximately 93,000 existing species (Bennett & Turgeon, 2017). Members of Ascomycota are ubiquitous and distributed across terrestrial, freshwater, and marine ecosystems (Bennett & Turgeon, 2016). Sexual reproduction in ascomycetes frequently occurs in response to adverse environmental conditions, leading to significant genetic diversity among species (Nieuwenhuis & James, 2016; Agrawalet al., 2018). The purpose of purging deleterious mutations and selecting advantageous mutations for adaptation to fluctuating environments (Otto & Lenormand, 2002).

Preussia (Sporormiaceae, Pleosporales, Dothideomycetes) was proposed by Fuckel (1866) to include species of ascomycetes with non-ostiole ascomycota, bitunicate asci and multi-celled ascospores with a germ slit in each cell and a gelatinous hyaline sheath, inhabiting mainly soil and fallen dead leaves, while anamorphs is usually represented by Chrysosporium spp. and Phoma spp. (Von Arx, 1973; Asgari & Zare, 2010; Crouset al., 2018; Lückinget al., 2020). Recently, the Index of Fungorum (2020) has listed 98 species of Preussia. In Iraq, many new species belonging to this genus were isolated, most of which were isolated from dung and plant debris (Guarroet al., 1997a, Guarroet al., 1997b, Abdullahet al., 1999) in addition, Preussia dispersa was also isolated from the marshes of southern Iraq by Abdullahet al. (2010).

Materials and Methods

Sample Collection

The sample of sedimentary soil was collected from the seacoast sediment of the Al-Faw region during August 2021, while the salinity and pH of the soil sample were calculated by salinometer (Lovibond, Germany) and pH meter (Adwa, Romany), respectively.

Two different types of media, Potato carrot agar (PCA) and Potato dextrose agar (PDA) (Himedia), were used for the isolation of fungus isolate by using the dilution method described by Wicklow and Whittingham (1974) culture media were incubated at 25 °C for two weeks. After incubation period, fungal growth was examined and purified then pure colonies were kept in PDA slant at 4 °C.

Identification Study

The fungal isolate was identified morphologically according to the appearance of colonies in addition to microscopic properties.

Molecular Identification and Phylogenetic Analysis

Genomic DNA extraction of the X6 isolate, primers synthesis, PCR amplification, and sequencing were conducted at Macrogen Company, South Korea. The universal primers ITS5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) were used to amplify the ITS gene encoding the 5.8S rRNA (ITS1-5.8SrRNA-ITS2 fragment) (Whiteet al., 1990). For further identification TEF1α primers were used through amplification the translational elongation factor 1α (TEF1α) using primers EF1–1018F (5′-GAY TTC ATC AAG AAC ATG AT-3′) and EF1–1620R (5′-GAC GTT GAA DCC RAC RTT GTC-3′). Gradient PCR was applied ranging from (48 °C–68 °C). The best annealing Temp was 48 °C, as shown in Fig. 1. The sequence results were analysed by BLAST, Basic Local Alignment Search Tool, and compared with the database at NCBI, the National Centre for Biotechnology Information (Altschulet al., 1997). The sequences were deposited to Gen Bank for accession Number confirmation. Alignments for each data set were made by MEGA11 using the ClustalW algorithm and refined with MUSCLE. The alignment comprised sequences from isolated species and those representing various species within the genus of Preussia, sourced from the NCBI database. Sequence phylogeny was constructed employing Maximum Likelihood (ML) and the most suitable nucleotide substitution model determined by MEGA11. Models with the lowest Bayesian Information Criterion (BIC) scores are regarded as the most accurate descriptors of the substitution pattern. The bootstrap consensus tree was inferred from 1000 replicates. After genomic analysis, the sequence data of isolated endophytic fungi were submitted to GenBank for Accession Number verification.

Fig. 1. The best annealing temperature for TEF1α region amplification.

Result of Phylogeny

The PCR amplification of the ITS region and TEF from isolate X6 produced PCR products measuring 500 and 567 base pairs (bp), respectively. For species identification, the sequences generated in this study were aligned against all sequences of well-documented Preussia species. The outcome of BLAST searching, indicating that the isolated species X6 has 88.58% similarity (ITS) with Preussia sp. (in: Fungi) isolate CK1259 (MH473918.1) and 96.72% similarity (TEF) with Preussia sp. (in: Fungi) strain SEED1 (KX894788.1). Thus, this isolated species was identified as a new species of Preussia genus and was named as Preussia aseelix. The gene bank accession numbers for X6 (ITS) and (TEF) were (OP214773) and (OP974691) respectively were confirmed and given by NCBI. This fungal species was reported first time in Basrah, IRAQ as the current study showed. Phylogenetic trees were constructed using DNA sequences from the isolated species in conjunction with reference strains. The resulting phylogenetic tree revealed a discernible relationship between the isolated species and the reference strains (Figs. 2A and 2B).

Fig. 2. Phylogenetic tree represents neighbour-joining analysis of (A) ITS domain and (B) TEF1α gene sequences depicting the relationships of the isolated fungi (isolate_X6) with closely related reference sequences of Preussia species retrieved from NCBI. Each numerical value represents the percentage of bootstrap samples, a total of 1000 samples, that support the internal branches with a confidence level of 50% or higher.

Physiological Assays

The physiological assays included measuring the growth at different temperatures (25 °C, 28 °C, and 37 °C) on PDA, as well as the test of cycloheximide resistance was evaluated.

Results and Discussion

The present finding revealed the isolation of Preussia aseelix as a new species of the world from sediment samples collected from southern Iraq. The salinity of samples was reached 51.3 ppt, whereas the acidity recorded at 8.12, and the sediment temperature was 30 °C.

Growth Condition

The optimum temperature for growth was 25 °C, in spite of the growth was failed at 37 °C. On the other hand, the isolate showed resistance of cycloheximide.

Taxonomy

Based on morphological notes and outcomes of molecular information and phylogenetic analysis, we erect the new species as follows:

• Preussia aseelix Almusa, Al-Maqtoofi, Alrubayae, and Altooma, sp.nov.
• Etymology: Named based on the first name of the author.
• Teleomorph undetermined. Anamorph coelemycetous. Vegetative hyphae subhyaline, thick walled, septate, branched, smooth walled (2.4–3.8 µm × 10.5–13 µm) wide. Conidiomata pycnidial, superficial or partly immersed, dark brown to black, globose to ellipsoidal or pyriform, solitary or aggregated 12.2–17 µm, with a narrow ostiole, pycnidial wall composed of hyaline to brown cells (2.6–4.3 µm × 2.6–4.7 µm). The external appearance of the pycidial wall is often coated with setae that seen as ramify scattered and straight, soft, varied from light to dark brown, septate (2.3–2.42 µm × 9.3–12.5 µm), with sharpened apex. Conidiophores are reduced to conidiogenous cells. Conidiogenous cells phialidic, hyaline, smooth, ampuliform to doliiform, conidia unicellular, hyaline, ampuliform, cylindrical, straight or slightly curved, aseptate, occasionally allantoids, often biguttulate, 1.15–1.84 µm × 2.9–4 µm. Chlamydospores globose to subglobose, unicellular, arranged intercalary or terminal, occasionally polygonal, brown to olivaceous in color, always found in the chain (6.7–8.2 × 7.3–11 µm).
• Culture characteristics: colonies on PDA attaining 30–40 mm diameter in two weeks at 25 °C, downy, floccose, grey becoming dark, deeply immersed into the agar, crinkled, raised in the centre and flat at the edges, with regular margins, reverse yellowish brown. Meanwhile, colonies on PCA with a diameter of 30–35 mm in two weeks at 25 °C, forming concentring rings composed of pycnidia, flat with buff shades at the edges, margins regular, and reverse of the same colour (Fig. 3).

Fig. 3. The morphological feature of the isolated species, Preuassia aseelix: (A) Colony on PDA after 14 days; (B) Colony on PCA after 14 days; (C) pycnidial conidiomata; (D) Setae; (E) Pycnidial wall; (F) Conidia; (G,H) Chlamydospores; (I) Vegetative hyphae.

Similarly, a new species of Preuassia named Preussia mediterranea was recorded from an endophyte of different typical Mediterranean plant species, which was described and illustrated based on molecular and morphological features. The new species Preussia mediterranea resembles P. australis, P. africana and P. similis from a morphological point of view, but appeared closer to P. minima and P. isabellae by molecular analysis (Arenalet al.,2007).

Interestingly, the present isolate resembles another new species, Preuassia aegilopis, that was isolated as an endophytic species from seeds of Aegilops umbellulata in Iran by not having a real fruiting body and being limited to the asexual phase only but differed with its morphologically and genetically (Mehrabiet al.,2022).

Finally, the current investigation showed a description of novel Preussia species depending on morphological features and phylogenetic analysis which drawn from sequence analysis of the ITS region that does not give convincing results, while TEF1α region confirms the identification of new species.