Macroalgal Endophytes from the Atlantic Coast of Canada: A Potential Source of Antibiotic Natural Products?

As the need for new and more effective antibiotics increases, untapped sources of biodiversity are being explored in an effort to provide lead structures for drug discovery. Endophytic fungi from marine macroalgae have been identified as a potential source of biologically active natural products, although data to support this is limited. To assess the antibiotic potential of temperate macroalgal endophytes we isolated endophytic fungi from algae collected in the Bay of Fundy, Canada and screened fungal extracts for the presence of antimicrobial compounds. A total of 79 endophytes were isolated from 7 species of red, 4 species of brown, and 3 species of green algae. Twenty of the endophytes were identified to the genus or species level, with the remaining isolates designated codes according to their morphology. Bioactivity screening assays performed on extracts of the fermentation broths and mycelia of the isolates revealed that 43 endophytes exhibited antibacterial activity, with 32 displaying antifungal activity. Endophytic fungi from Bay of Fundy macroalgae therefore represent a significant source of antibiotic natural products and warrant further detailed investigation.

1. Introduction

The threat to human health as a result of the growing emergence of microbial resistance to antibiotic agents is both real and significant; current forecasts predict that broad-scale antimicrobial ineffectiveness is imminent and we may soon once again face the problems that challenged medicine in the “pre-antibiotic” era [1,2,3]. There is, therefore, an urgent need to accelerate the discovery of antibiotic molecules to facilitate the development of new therapeutic agents with novel modes-of-action to combat infectious disease [4,5]. Endophytes of macroalgae have recently gained attention as an untapped source of biodiversity with potential to yield novel bioactive metabolites [6,7,8] and high proportions of isolates from both tropical [9] and temperate [10,11] environments have exhibited significant antibiotic activities. However, data relating to the bioactivity of endophyte assemblages obtained from a given algal species is limited; evaluating the true potential of macroalgal endophytes as a source of antibiotic compounds is therefore problematic. Recent reports also suggest that there may be significant differences in the endophyte assemblages found within tropical and temperate marine macroalgal hosts [9,11], although further work is required to confirm these preliminary observations. The objectives of this study were to perform a preliminary investigation of the endophytes from macroalgae from the Atlantic coast of Canada and evaluate their potential for the production of antimicrobial compounds.

2. Experimental Section

2.1. Algal Collection

Fourteen species of algae were collected from L’Etete, New Brunswick, Canada (45° 02.372′ N, 66° 53.424′ W) from August to November 2009. Seven species of red algae (Porphyra umbilicalis, Porphyra purpurea, Palmaria palmata, Chondrus crispus, Devaleraea ramentacea, Mastocarpus stellatus and Polysiphonia lanosa), four species of brown algae (Fucus spiralis, Fucus vesiculosus, Saccharina latissima and Ascophyllum nodosum) and three species of green algae (Spongomorpha arcta, Ulva intestinalis and Ulva lactuca) were investigated for the presence of endophytic fungi. In all cases algae with no obvious signs or symptoms of disease were used.

2.2. Surface Sterilization of Algae and Culture Techniques

The surfaces of the algal samples were sterilized by immersion in various sterilant solutions [11]. Prior to the isolation of endophytic fungi from marine algae, an optimal surface sterilization method was developed for each algal species (Table 1). Portions (5 cm2) of algal tissue were individually surface sterilized using the appropriate optimized technique, blotted dry on autoclaved paper towel and rubbed across the surface of 2 plates of 2% malt extract agar (MEA, Becton Dickinson, Sparks, MD, USA) prepared with artificial seawater (MEA-SW, 24.4 g·L−1; Instant Ocean® Sea Salt, Cincinnati, OH, USA) to verify surface sterilization had been effective. Sterilized algal species were cut using a sterile cork borer and placed on Petri plates of 2% MEA and 2% MEA with seawater. Petri plates were sealed with Parafilm™ (Pechiney Plastic Packaging Company, Chicago, IL, USA) prior to incubation.

Table 1

Surface sterilization techniques used on marine algae collected from the Bay of Fundy, Canada. 1

Algal Species	Sterilant Immersion Duration (Seconds)
	Bleach (5.25%)	Ethanol (70%)
Chondrus crispus	5	10
Devaleraea ramentacea	5	10
Mastocarpus stellatus	5	15
Palmaria palmata	0	15
Polysiphonia lanosa	0	10
Porphyra purpurea	0	10
Porphyra umbilicalis	0	10
Ascophyllum nodosum	0	15
Fucus spiralis	0	15
Fucus vesiculosus	0	15
Saccharina latissima	5	15
Spongomorpha arcta	0	20
Ulva intestinalis	0	20
Ulva lactuca	0	20

1 All surface sterilizations were confirmed by the absence of microbial colony formation on 2 surface verification plates (2% malt extract with artificial seawater).

2.3. Isolation of Endophytes

Petri plates containing surface sterilized macroalgal pieces were incubated for 14 days at room temperature (approximately 25 °C) under ambient light conditions and monitored daily for the presence of hyphae growing from the cut edges of the algal segments. The isolation frequency (IF) of emerging hyphae was determined for each species of algae collected [12]: Endophytes growing from the cut edges of the segments were subcultured onto fresh media (2% MEA) to obtain pure isolates. Pure isolates were also grown on Czapek, potato dextrose, cornmeal and marine agars (Becton Dickinson, Sparks, MD, USA) to induce sporulation and differentiate between colony morphologies. Individual isolates from each algal species were then sorted into groups of homogeneous morphotypes, and a representative colony of each distinct isolate was used for the fungal identification, fermentation, and extraction.

2.4. Identification of Fungi

Fungal isolates were identified taxonomically through examination of colony and spore morphology with taxonomic classifications being confirmed by comparison of the internal transcribed spacer and 5.8S rRNA gene (ITS) DNA regions [13] with corresponding sequences available in the GenBank database (National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, MD, USA).

The genomic DNA of all distinct fungal isolates was extracted using a DNeasy® plant mini kit (Qiagen, Toronto, Ontario, Canada) and amplified by PCR as previously described [11]. Samples of amplified ITS DNA were submitted for sequencing (Génome-Québec; Montreal, Québec, Canada) along with the ITS 1 and ITS 4 primers. The sequences obtained were checked for ambiguity and submitted to the GenBank database and compared with existing GenBank sequence data using BLAST. In cases where electrophoresis indicated that the DNA extraction/ PCR procedure had been unsuccessful, the procedure was repeated on a fresh sample of the fungal isolate.

Isolates identified taxonomically to species level had ≥99% sequence similarities to entries for conspecifics in GenBank, isolates identified to the genus level had ≥96% sequence similarities with congeneric species, and isolates identified to class level had sequence similarities ≥82% to entries from the corresponding taxonomic rank. Isolates that could not be unequivocally identified to species level based on morphological observations were only classified to the corresponding genus even when the sequence similarities ≥98% were obtained with sequence data for congeneric species in GenBank. If DNA from the ITS region was not isolated after three extraction/amplification attempts, the corresponding isolate was identified on morphological observations alone. Sterile isolates that did not provide sequence data upon repeated attempts were given codes according to their morphology in plate culture [9,14,15]. All distinct fungal isolates have been archived in the UNB Saint John fungal repository (Saint John, New Brunswick, Canada).

2.5. Preparation of Extracts

For each fungal isolate, a portion (5 mm2) of the fungal colony on solid medium was transferred to a 250 mL Erlenmeyer flask containing 2% Bacto™ malt extract broth (100 mL). Flasks were shaken (150 rpm) at room temperature under ambient light conditions for two weeks.

After incubation, the fungal mycelia from each culture were separated from the spent culture broth using vacuum filtration. Mycelia were extracted once with methanol (50 mL; Fisher Scientific, Ottawa, Ontario, Canada) in the dark for 24 h at 4 °C, solid residue and cell debris were removed by vacuum filtration, and the resulting solution was concentrated in vacuo to give a crude fungal extract. The spent culture broths were extracted 3 times with 50 mL ethyl acetate (Fisher Scientific) and the combined organic extracts were concentrated in vacuo to give a crude extract of the growth media. All crude extracts were stored at −20 °C until required.

2.6. Antibacterial and Antifungal Activity Assay

Antibacterial and antifungal activity against Pseudomonas aeruginosa (ATCC 10145), Staphylococcus aureus (ATCC 29213) and Candida albicans (ATCC 14053) was evaluated using a microbroth dilution antimicrobial susceptibility assay as previously described [11] with extracts being tested at a concentration of 200 μg·mL−1.

2.7. Statistical Analyses

Antimicrobial activities of extracts were compared to the negative control using the Kruskal-Wallis non-parametric test as the data was not normally distributed (Shapiro-Wilk, p < 0.05) and the variances were not equal (Levene’s test, p < 0.05). Post hoc analysis (stepwise-stepdown) was performed to determine which extracts differed from the negative control. Crude extracts were defined as biologically active if their effect on the growth of the test organism was significantly different (p < 0.05) to the negative control (0% inhibition). All statistical tests were performed using SPSS (PASW Statistics 18, IBM Corporation, Armonk, NY, USA).

3. Results and Discussion

3.1. Isolation of Endophytic Fungi from Marine Algae

The Bay of Fundy, with its extreme tidal range, presents a unique temperate habitat and supports a high diversity of marine macroalgal species [16,17], many of which have not been investigated for the presence of endophytes. Our results indicate that these algae support a sizeable and diverse assemblage of endophytes: fungi were isolated from 632 out of a total 3081 algal pieces resulting in an overall isolation frequency of 26% (Table 2). The isolation frequencies of endophytic fungi varied by host, with the highest isolation frequencies obtained from the red algae P. palmata and P. umbilicalis, at rates of 87% and 72% respectively (Table 2). The lowest isolation frequencies were from the red alga C. crispus at a rate of 0.08% (Table 2).

Table 2

Isolation frequencies and distinct number of isolates of endophytic fungi from marine algae collected from the Bay of Fundy, Canada.

Seaweed Species	Total Algae Segments	Pieces with Endophytic Growth	Isolation Frequency (%)	Distinct Fungal Taxa
Chondrus crispus	246	2	0.08	2
Devaleraea ramentacea	108	46	43	13
Mastocarpus stellatus	98	32	33	9
Palmaria palmata	167	146	87	7
Polysiphonia lanosa	67	12	18	5
Porphyra purpurea	85	17	20	2
Porphyra umbilicalis	217	156	72	3
Ascophyllum nodosum	152	16	11	1
Fucus spiralis	192	16	8	4
Fucus vesiculosus	109	6	6	1
Saccharina latissima	1138	95	8	13
Spongomorpha arcta	184	49	27	11
Ulva intestinalis	117	24	21	5
Ulva lactuca	201	15	8	3
Total	3081	632	26	79

Seventy-nine distinct endophyte species were isolated from the ten algal hosts (Table 3). The number of distinct fungal isolates obtained varied by host with a maximum of 13 endophytic fungal species from D. ramentacea and S. latissima to a minimum of one from A. nodosum and F. vesiculosus (Table 3). Penicillium spp. were isolated from seven algae: C. crispus, D. ramentacea, M. stellatus, P. palmata, S. latissima, S. arcta and U. lactuca with a total of 11 isolates representing the six species isolated (Table 3). Six distinct Aspergillus spp. were isolated from five algae species: C. crispus, P. umbilicalis, A. nodosum, S. latissima and U. intestinalis (Table 3). Botrytis sp. was isolated from two algal hosts (D. ramentacea and P. palmata; Table 3) as was Aureobasidium pullulans (D. ramentacea and P. lanosa; Table 3). Cladosporium sp., Trametes versicolor, Coniothyrium sp., Coelomycete I, Hypoxylon sp., Helicomyces sp. and Botryotinia fuckeliana were only isolated from one host (Table 3).

Table 3

Identification of endophytic fungi isolated from marine algae collected from the Bay of Fundy.

Host species	Isolate	Species	Accession number
Chondrus crispus	AF8-053	Aspergillus sp. I	KF572134
	AF8-129	Penicillium crustosum I	KF572146
Devaleraea ramentacea	AF8-079	Cladosporium sp.	KF572136
	AF8-081	Septate Pigmented I	-
	AF8-083	Septate Pigmented II	-
	AF8-085	Sterile Hyaline I	-
	AF8-087	Sterile Hyaline II	-
	AF8-089	Botrytis sp. I	-
	AF8-091	Penicillium decumbens I	KF572138
	AF8-093	Septate Pigmented III	-
	AF8-095	Aureobasidium pullulans I	-
Devaleraea ramentacea	AF8-109	Trametes versicolor	KF572133
	AF8-111	White Fluffy II	-
	AF8-113	Coniothyrium sp.	KF572141
	AF8-115	Botrytis sp. II	-
Mastocarpus stellatus	AF8-055	Penicillium sp.	-
	AF8-057	Red Yeast I	-
	AF8-059	Septate Pigmented IV	-
	AF8-061	Red Yeast II	-
	AF8-063	Sterile Hyaline III	-
	AF8-065	Sterile Hyaline IV	-
	AF8-067	Coelomycete I	-
	AF8-101	Penicillium decumbens II	KF572139
Palmaria palmata	AF8-069	Sterile Hyaline V	-
	AF8-071	Hypoxylon sp.	KF572135
	AF8-073	Penicillium decumbens III	KF572149
	AF8-075	Penicillium chrysogenum I	KF572137
	AF8-077	Helicomyces sp.	-
	AF8-103	Penicillium crustosum II	KF572147
	AF8-105	Botrytis sp. III	-
	AF8-107	Sterile Hyaline VI	-
	AF8-123	Sterile Hyaline VII	-
Polysiphonia lanosa	AF8-097	Sterile Hyaline VIII	-
	AF8-099	Aureobasidium pullulans II	KF572140
	AF8-117	Sterile Hyaline IX	-
	AF8-119	Botryotinia fuckeliana	KF572148
	AF8-121	Sterile Hyaline X	-
Porphyra purpurea	AF8-125	Sterile Hyaline XI	-
	AF8-127	Sterile Hyaline XII	-
Porphyra umbilicalis	AF8-049	Sterile Hyaline XIII	-
	AF8-051	Sterile Hyaline XIV	-
	AF8-131	Aspergillus sydowii	KF572145
Ascophyllum nodosum	AF1-141B	Aspergillus sp. II	-
Fucus spiralis	AF1-021A	Black Hyaline I	-
	AF1-021B	Septate Pigmented V	-
	AF1-021D	Pigmented Hyaline I	-
	AF1-021G	Sterile Hyaline XV	-
Fucus vesiculosus	AF2-059A	Sterile Hyaline XXI	-
Saccharina latissima	AF1-029A2	White Hyaline IV	-
	AF1-029B2	Black Hyaline II	-
	AF1-073C	Penicillium chrysogenum II	KF572144
	AF1-073D	Aspergillus sp. III	-
	AF1-073E	White Hyaline I	-
	AF1-073G	Sterile Beige I	-
	AF1-073N	Sterile Beige II	-
	AF2-055B	Penicillium soppii I	KF572151
	AF2-055F	Sterile Hyaline XVI	-
	AF2-055G	White Hyaline II	-
	AF2-055O	Penicillium chrysogenum III	KF572150
	AF2-055P	Pigmented Hyaline IV(Red)	-
Spongomorpha arcta	AF1-037C2	Sterile Hyaline XVII	-
	AF1-037F	Sterile Beige III	-
	AF1-153A	Pigmented Hyaline VII	-
	AF1-153B	Sterile Hyaline XVIII	-
	AF1-153C	White Hyaline III	-
	AF1-153D	Penicillium spinulosum	KF572143
	AF1-153H	Black Hyaline III	-
	AF1-153J	Pigmented Hyaline VI	-
	AF1-153L	Pigmented Hyaline V	-
	AF1-153M	Penicillium sp.	KF572152
	AF1-153N	Penicillium soppii II	-
Ulva intestinalis	AF2-063C	Sterile Hyaline XIX	-
	AF2-063E	Septate Pigmented VIII	-
	AF2-063F	Pigmented Hyaline VIII	-
	AF2-063G	Aspergillus sp. V	-
	AF2-063H	Septate Pigmented VI	-
Ulva lactuca	AF1-033A2	Septate Pigmented VII	-
	AF1-033B	Sterile Hyaline XX	-
	AF1-033C	Penicillium chrysogenum IV	KF572142

The majority of isolates (74%), however, could neither be identified morphologically nor through the use of molecular genetic techniques. These isolates were designated codes according to their plate morphology (Table 3). This proportion of sterile mycelia (74%) is high in comparison to other marine macroalgal endophytes from the North Atlantic (45%) [11] and may have contributed to a correspondingly lower success rate observed for the molecular identification of isolates in this study (26% compared with 55% for endophytes isolated from macroalgae of the Shetland Islands, UK) [11]. The magnitude of these discrepancies is surprising and may be due to the particular assemblage of fungal species isolated from the Bay of Fundy algae. Further work will be required to optimize procedures used for molecular identification of sterile algal endophytes in an effort to increase our ability to identify fungi isolated from this source.

Penicillium spp. and Aspergillus spp. are common endophytes of marine macroalgae [9,11], and represent nearly one quarter (24%, 19/79 isolates) of the distinct isolates obtained from macroalgae of the Bay of Fundy. The majority of fungi obtained from Bay of Fundy macroalgae were isolated as sterile mycelia and several of the endophytes, i.e., Botrytis spp., Hypoxylon sp., and Helicomyces sp., have not been previously isolated from marine algal hosts. Many of the macroalgae investigated in this study, S. latissima, S. arcta, P. purpurea, P. umbilicalis, P. palmata, M. stellatus and D. ramentacea, have not been previously investigated for their endophytic fungi. Endophytes have previously been reported from A. nodosum, F. spiralis, F. vesiculosus, P. lanosa, U. intestinalis and U. lactuca [9,11,18,19,20,21,22,23,24,25,26,27,28,29,30]. However, there was a high degree of variability in the diversity of fungi obtained in these studies, which suggests limited host specificity of these fungal endophytes to their hosts.

A point of particular interest is the fact that none of the fungi isolated from the brown alga A. nodosum were identified as Mycosphaerella ascophylli, despite it being a well documented endophyte of that host [18,19,20,21,22,24,27,28,30]. Whilst it is possible that M. ascophylli may not have been isolated due to the particular sterilization and culture conditions used or the low growth rate of the fungus [19], it is more probable that M. ascophylli is represented in the mycelia sterilia obtained from A. nodosum as it is known to display a sterile morphotype of fine white septate hyphae [19,31].

3.2. Antimicrobial Screening Results of Algal-Derived Fungal Endophytes

Crude extracts of the endophyte isolates were screened against three pathogenic microorganisms, the Gram positive bacterium Staphylococcus aureus, the Gram negative bacterium Pseudomonas aeruginosa and the fungus Candida albicans. Extracts were defined as bioactive if they inhibited growth of the test organism in comparison to the negative control as indicated by heterogeneous subsets (p < 0.05) identified through post hoc testing of Kruskal-Wallis analyses. Seventy-eight crude extracts, tested at 200.0 μg·mL−1, inhibited at least one of the test microorganisms. Forty-four crude extracts significantly inhibited the growth of S. aureus, whereas, 18 and 36 extracts inhibited the growth of P. aeruginosa and C. albicans, respectively. These extracts were derived from 57 individual fungal isolates (Table 4) and comprised 39 mycelia-derived and 39 medium-derived crude extracts. Of the 156 crude extracts tested, 59 showed activity against only 1 test microorganism, 18 inhibited 2 microorganisms, and 1 showed activity against all 3 test microorganisms (Table 4). In this study, 73% of the isolates (57/78) were found to have antimicrobial activity against P. aeruginosa, S. aureus, and C. albicans (43/78, 55% antibacterial; 32/78, 41%, antifungal). The antimicrobial screening results of this study are comparable to those obtained from marine algae of the Shetland Islands, UK where 61% of the isolates (39/64) had activity against at least one of the same three test organisms (36/64 isolates antibacterial and 24/64 isolates antifungal) [11]. However, these antimicrobial screening results are in contrast to those obtained from endophytic fungi of macroalgae of the Southern Indian coast (82%, 31/38 isolates) [9] and the coasts of the North and Baltic Seas (83%, 249/300) [10]. The crude extracts showing strong antimicrobial bioactivity in this work should be investigated to identify the biologically active constituents responsible for the observed bioactivity.

Table 4

Activity of crude extracts showing significant inhibition to S. aureus, P. aeruginosa and C. albicans, obtained from endophytic fungi isolated from marine algae collected from the Bay of Fundy.

Fungal Taxa	Host Alga	Extract 1 Source	Inhibition (%) 2
			SA 3	PA 3	CA 3
Aspergillus sp. I	C. crispus	Mycelia	-	18 ± 1	-
Aspergillus sp. I	C. crispus	Media	28 ± 3	-	15 ± 3
Penicillium crustosum I	C. crispus	Media	43 ± 1	-	-
Cladosporium sp.	D. ramentacea	Mycelia	36 ± 1	9 ± 1	-
Septate Pigmented I	D. ramentacea	Mycelia	-	-	17 ± 2
Sterile Hyaline I	D. ramentacea	Mycelia	-	20 ± 1	-
Sterile Hyaline I	D. ramentacea	Media	29 ± 2	-	-
Botrytis sp. I	D. ramentacea	Mycelia	94 ± 1	-	-
P. decumbens I	D. ramentacea	Mycelia	36 ± 4	-	98 ± 1
P. decumbens I	D. ramentacea	Media	-	-	99 ± 1
Septate Pigmented III	D. ramentacea	Mycelia	47 ± 4	-	-
Aureobasidium pullulans I	D. ramentacea	Media	82 ± 4	51 ± 2	13 ± 2
Trametes versicolor	D. ramentacea	Mycelia	-	17 ± 2	-
White Fluffy II	D. ramentacea	Media	-	-	12 ± 1
Coniothyrium sp.	D. ramentacea	Mycelia	31 ± 1	-	-
Coniothyrium sp.	D. ramentacea	Media	25 ± 2	28 ± 2	-
Botrytis sp. II	D. ramentacea	Mycelia	-	-	14 ± 1
Botrytis sp. II	D. ramentacea	Media	97 ± 1	-	-
Red Yeast I	M. stellatus	Mycelia	31 ± 2	-	-
Septate Pigmented IV	M. stellatus	Mycelia	-	16 ± 3	-
Septate Pigmented IV	M. stellatus	Media	23 ± 1	-	70 ± 3
P. decumbens II	M. stellatus	Media	-	-	12 ± 1
Sterile Hyaline V	P. palmata	Mycelia	-	11 ± 1	15 ± 4
Sterile Hyaline V	P. palmata	Media	-	-	16 ± 1
Hypoxylon sp.	P. palmata	Mycelia	-	13 ± 3	30 ± 3
Hypoxylon sp.	P. palmata	Media	-	-	18 ± 2
P. decumbens III	P. palmata	Mycelia	-	-	16 ± 5
P. chrysogenum I	P. palmata	Media	58 ± 3	-	-
Helicomyces sp.	P. palmata	Mycelia	21 ± 1	-	-
Helicomyces sp.	P. palmata	Media	-	-	21 ± 5
P. crustosum II	P. palmata	Mycelia	82 ± 5	-	22 ± 3
P. crustosum II	P. palmata	Media	39 ± 2	-	-
Botrytis sp. III	P. palmata	Media	92 ± 1	-	-
Sterile Hyaline VI	P. palmata	Mycelia	-	-	13 ± 2
Sterile Hyaline VI	P. palmata	Media	19 ± 6	-	-
Sterile Hyaline VII	P. palmata	Media	90 ± 2	-	24 ± 2
Aureobasidium pullulans II	P. lanosa	Mycelia	49 ± 1	34 ± 3	-
Sterile Hyaline IX	P. lanosa	Media	-	-	40 ± 5
Botryotinia fuckeliana	P. lanosa	Mycelia	96 ± 1	-	30 ± 2
Botryotinia fuckeliana	P. lanosa	Media	41 ± 2	-	-
Sterile Hyaline X	P. lanosa	Mycelia	-	-	20 ± 11
Sterile Hyaline XI	P. purpurea	Mycelia	22 ± 1	-	-
Sterile Hyaline XI	P. purpurea	Media	34 ± 9	-	-
Sterile Hyaline XII	P. purpurea	Mycelia	50 ± 1	-	-
Sterile Hyaline XIII	P. umbilicalis	Media	-	-	33 ± 1
Sterile Hyaline XIV	P. umbilicalis	Mycelia	-	14 ± 1	27 ± 6
Sterile Hyaline XIV	P. umbilicalis	Media	22 ± 5	-	-
Aspergillus sydowii	P. umbilicalis	Media	51 ± 3	-	-
Aspergillus sp. II	A. nodosum	Mycelia	66 ± 4	-	-
Aspergillus sp. II	A. nodosum	Media	85 ± 1	-	15 ± 3
Black Hyaline I	F. spiralis	Media	41 ± 2	-	-
Septate Pigmented V	F. spiralis	Mycelia	39 ± 3	8 ± 1	-
Septate Pigmented V	F. spiralis	Media	27 ± 3	-	-
Pigmented Hyaline I	F. spiralis	Mycelia	53 ± 4	-	-
White Hyaline IV	S. latissima	Media	-	-	30 ± 1
Black Hyaline II	S. latissima	Mycelia	-	-	13 ± 5
P. chrysogenum II	S. latissima	Mycelia	25 ± 4	-	-
P. chrysogenum II	S. latissima	Media	31 ± 4	-	-
Aspergillus sp. III	S. latissima	Mycelia	25 ± 1	-	-
Sterile Beige II	S. latissima	Media	-	-	14 ± 3
White Hyaline II	S. latissima	Mycelia	28 ± 3	-	27 ± 5
White Hyaline II	S. latissima	Media	21 ± 1	-	18 ± 9
Pigmented Hyaline IV(Red)	S. latissima	Mycelia	-	12 ± 1	-
Pigmented Hyaline IV(Red)	S. latissima	Media	-	-	17 ± 1
Sterile Hyaline XVII	S. arcta	Mycelia	-	-	13 ± 1
Pigmented Hyaline VII	S. arcta	Media	-	15 ± 3	-
White Hyaline III	S. arcta	Mycelia	27 ± 7	-	-
White Hyaline III	S. arcta	Media	-	-	13 ± 3
Penicillium spinulosum	S. arcta	Mycelia	65 ± 4	-	-
Penicillium spinulosum	S. arcta	Media	37 ± 1	-	-
Pigmented Hyaline V	S. arcta	Media	87 ± 3	-	82 ± 1
Septate Pigmented VIII	S. arcta	Mycelia	-	17 ± 1	-
Septate Pigmented VIII	S. arcta	Media	36 ± 2	-	-
Penicillium soppii II	S. arcta	Media	-	9 ± 3	-
Sterile Hyaline XIX	U. intestinalis	Media	-	9 ± 2	22 ± 2
Septate Pigmented VI	U. intestinalis	Mycelia	-	-	13 ± 2
Septate Pigmented VII	U. lactuca	Mycelia	-	-	18 ± 2
Sterile Hyaline XX	U. lactuca	Mycelia	-	24 ± 3	-

1 Crude extracts were tested at 200.0 μg·mL−1; activity is defined as microbial inhibition significantly differing from negative control; 2 Percentage inhibition is represented as the mean of 3 readings ± SE; 3 SA: Staphylococcus aureus; PA: Pseudomonas aeruginosa; CA: Candida albicans.

4. Conclusions

Our research has demonstrated that marine macroalgae from the Bay of Fundy, Canada, have the potential to be an excellent source of endophytic fungi. Seventy-eight distinct isolates were obtained from 14 algal hosts, with most of the endophytes that could be taxonomically identified belonging to the genera Penicillium and Aspergillus. The results from the antimicrobial screening on the mycelium and broth extracts of the endophytic fungi suggest that they are a promising source of antimicrobial extracts, with 18 extracts exhibiting >50% inhibition in the screening assays. These crude extracts should be subjected to bioassay-guided fractionation in an attempt to identify the bioactive constituents of the extracts. Further work should also be focused on improving the rate of identification either through the use of molecular techniques, as only 26% of the obtained isolates were identified through this method, or through the induction of conidia or other distinguishing morphological characteristics. The results from this study has led to further work into the endophytic fungi of Atlantic coast marine macroalgae, with a current investigation now focused on the isolation, identification and antimicrobial screening of endophytic fungi from the diverse range of macroalgae present in the Bay of Fundy.