Ulva sp.

Noor, Sadia, Begum, Mst. Nadira, Rony, Satyajit Roy, Uddin, Mohammad Zashim, Sohrab, Md. Hossain & Mazid, Md. Abdul, 2024, Bioactivity and chemical screening of endophytic fungi associated with the seaweed Ulva sp. of the Bay of Bengal, Bangladesh, Botanica Marina (Warsaw, Poland) 67 (2), pp. 115-129 : 118-120

3.2 Endophytic fungi associated with Ulva sp.

The isolation and identification of fungal endophytes from Ulva sp. revealed six different fungi that were most closely related to Chaetomium globosum , Nigrospora magnoliae , Curvularia sp. , Curvularia moringae , Aspergillus terreus and Collariella sp. (Table 1).

The isolate UE-1 ( Figure 1 ) was a rapidly growing endophytic fungus. The surface of the colony was cottony and white initially, becoming greyish-olive with age. The reverse of the colony was yellowish to reddish brown. The colony diameter on PDA was approximately 7.8–8.0 cm at 28 ± 2 ° C after 6 days in culture. Hyphae were septate with large, ovalshaped, brown-coloured ascomata with wavy filamentous hairs. Asci were stalked and club-shaped, containing ascospores. Ascospores were limoniform, single-celled and yellowish-brown. The colony morphology and microscopic observations suggested the identity of the isolate UE-1 to be a species of Chaetomium (Wang et al. 2016) , which was confirmed by the nucleotide BLAST report generated by NCBI database (76.4 % similarity to BLAST best hit with 100 % query coverage). Chaetomium globosum (Accession no. NR 144851.1, connected with the marine environment) was discovered in the phylogenetic tree to be the closest to the isolate UE-1, with a bootstrap support of 92 %. Thus, the isolate UE-1 was recognized as Chaetomium globosum , and its morphology matched that of the relevant species.

The endophytic fungus UE-2 was initially white, becoming grey with abundant aerial mycelia ( Figure 2 ). Black areas of conidiation appeared with age. Initially, the reverse of the colony was white and gradually became black. Colonies on PDA reached 7.5–7.8 cm diameter after 6 days at 28 ± 2 ° C. Mycelia were superficial and immersed, composed of septate, branched, hyaline and brown hyphae with smooth and thick walls. Conidiophores were short, swelling and tapering at the point of conidium formation. The conidia were dark brown and almost round, slightly flattened. Morphological characteristics indicated that UE-2 was a species of Nigrospora genus ( de Silva et al. 2021). The nucleotide BLAST result showed 98.2 % similarity to its best hit with 90 % query coverage. Phylogenetic analysis revealed that this isolate was most closely related to Nigrospora magnoliae (Accession no. NR_172443.1, associated with terrestrial plants), sharing a monophyletic clade with a reasonably good bootstrap support of 85 % ( Hillis and Bull 1993). As a result of phylogenetic analysis and morphology, isolate UE-2 was identified as Nigrospora magnoliae when compared to other Nigrospora species.

The isolate UE-3 initially appeared as a pinkish colony, growing moderately and gradually turning black; the reverse was the same as the surface ( Figure 3A–C ). The colony diameter on PDA was approximately 4.5–5.0 cm at 28 ± 2 ° C after 6 days of culture. The colony was circular with an entire margin and a woolly texture. Hyphae were long, branched, septate and dark. At the site of conidium development, conidiophores were either simple and straight, branching and curved, or knobby. Conidia were big, typically had four cells, and ultimately took on a curved appearance from the enlargement of a central cell after 5–7 days of ageing. Conidia vary from those of Bipolaris sp. in that they have a darker central cell than the peripheral cells, a finer cell wall, thinner septa between cells, and a distinctive curve developed with maturity. These morphological characteristics indicated UE-3 as Curvularia sp. (Walsh et al. 2018).

The surface colour of isolate UE-4 was grey or black with a woolly surface ( Figure 3D–F ). The reverse of the colony was dark. The growth rate was moderate; the colony diameter on PDA was approximately 2.5–3.5 cm at 28 ± 2 ° C after 6 days of culture. Microscopic examination revealed mycelia and conidiophores with conidia, similar to those of isolate UE-3. Moreover, the nucleotide BLAST report generated from the ITS sequence of isolate UE-4 supported this assumption and showed 97.8 % similarity to BLAST best hit with 95 % query coverage. The phylogenetic tree clearly showed that UE-4 was closely related to Curvularia moringae (Accession no. NR_171998.1, associated with terrestrial plant) with a bootstrap value of 88 %, indicating a well supported monophyletic clade. Given these observations, UE-4 was specified as Curvularia moringae among the other genetically similar Curvularia species.

The isolate UE-5 was a filamentous endophytic fungus with a powdery texture ( Figure 4 ). Its growth rate was moderate, reaching 3.0– 3.5 cm diameter on PDA at 28 ± 2 ° C after 6 days of culture. The surface of the colony was at first white, and then a shade of yellow spreading from the centre and finally turned into cinnamon brown. The reverse of the colony was yellow to brown. Hyphae were septate along with short and smooth conidiophores. Phialides and metulae with chains of conidia were arranged solely on the top half of the vesicle, compact and column-like. Conidia were smooth and rounded. Microscopic and colony morphology indicated that the isolate was a species of Aspergillus (Walsh et al. 2018) . The nucleotide BLAST result showed 100 % similarity to its best hit with 96 % query coverage. In the phylogenetic tree, UE-5 had the closest evolutionary relationship with Aspergillus terreus (Accession no. NR_131276.1, associated with indoor dust), forming a monophyletic clade with a bootstrap support of 66 %. Thus, the molecular verification distinguished UE-5 as Aspergillus terreus among other Aspergillus species.

Colonies of the isolate UE-6 on PDA ( Figure 5A–C ) were translucent to off- white, with an entire or slightly undulate edge, about 3.0– 3.5 cm diameter at 28 ± 2 ° C after 6 days of culture, and irregularly produced white aerial hyphae and dark ascospores later; the reverse was the same as the surface view. Ascomatal hairs were brown, septate, relatively seta-like or flexuous. Ellipsoidal ascospores were brown. In phylogenetic analysis, UE-6 belonged to a monophyletic clade along with two highly similar species, Collariella gracilis and Collariella virescens , supported by a bootstrap value of 61 % and the best hits in the nucleotide BLAST with 88 % query coverage showed 94.3 % and 94.1 % similarity, respectively, to these species. The two Collariella species shared a 98 % bootstrap support value, as shown in Figure 5D . According to von Arx et al. (1986), these Collariella species are very similar, and differ only in ascomata and ascospores. Due to morphological and molecular similarities, these species were previously known as Chaetomium . Isolate UE-6 was phylogenetically identified as either Collariella gracilis or Collariella virescens (Accession no. NR_147670.1 and NR_147671.1, respectively, associated with soil and rock), whose morphology was also found to be similar to the isolate (Wang et al. 2016).

Figures

Figure 1: Isolate UE-1 (Chaetomium globosum). (A) Surface of colony, on potato dextrose agar after 6 days culture at 28 °C. (B) Reverse of colony.(C) Ascomata after 28 days culture. (D) Asci.(E) Ascus with ascospores.(F) Ascospores. (G) Phylogenetic tree inferred from internal transcribed spacer sequences using maximum likelihood method.

Figure 2: Isolate UE-2 (Nigrospora magnoliae). (A) Surface of colony, on potato dextrose agar after 6 days culture at 28 °C. (B) Reverse of colony. (C) Mycelia and conidia after 21 days culture.(D) Conidiogenus cells with conidia.(E) Phylogenetic tree inferred from internal transcribed spacer sequences using maximum likelihood method.

Figure 3: Isolates UE-3 (Curvularia sp.,A–C) and UE-4 (Curvularia moringae, D–H). (A) Surface of colony, on potato dextrose agar (PDA) after 6 days culture at 28 °C. (B) Reverse of colony. (C) Mycelia and conidia after 7 days culture.(D) Surface of colony,on PDA after 12 days culture at 28 °C. (E) Reverse of colony. (F) Mycelia and conidia after 7 days culture. (G) Conidium. (H) Phylogenetic tree inferred from internal transcribed spacer sequences using maximum likelihood method.

Figure 4: Isolate UE-5 (Aspergillus terreus). (A) Surface of colony, on potato dextrose agar after 6 days culture at 28 °C. (B) Reverse of colony.(C) Mycelia,conidiophores and conidia after 5 days culture. (D) Conidiophore with conidia. (E) Phylogenetic tree inferred from internal transcribed spacer sequences using maximum likelihood method.

Figure 5: Isolate UE-6 (Collariella sp.). (A) Surface of colony, on potato dextrose agar after 12 days culture at 28 °C. (B) Reverse of colony. (C) Terminal ascomatal hairs with ascospores after 45 days culture. (D) Phylogenetic tree inferred from internal transcribed spacer sequences using maximum likelihood method.