- freely available
Int. J. Mol. Sci. 2016, 17(1), 140; doi:10.3390/ijms17010140
Abstract: Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture.
Saprolegniosis, caused by Saprolegnia species, results in tremendous losses in wild and cultured fish species including salmonids such as salmon and trout, and non-salmonids such as tilapia, catfish, carp, and eel . The typical symptoms of Saprolegniosis are white or grey fungal-like hyphal mats on fish or their eggs . Yield losses of 10% to more than 50% have been reported in eggs and young fish [1,3,4].
To control Saprolegniosis, formalin is now commonly applied but is expected to be banned soon due to adverse effects on the environment . A limited number of chemical and non-chemical alternative treatments have been tested to control Saprolegniosis, including hydrogen peroxide, sea water flushes and ultraviolet irradiation, but none of these are as effective as the banned malachite green . Also, no vaccine is currently available to control this disease [1,5].
Bacterial genera such as Bacillus, Enterococcus and Lactobacillus have been shown to reduce specific diseases in aquaculture and several of these beneficial bacteria are being commercialized [6,7,8,9,10]. As a sustainable measure to combat Saprolegniosis, the bacterial genera Aeromonas, Frondihabitans and Pseudomonas have been proposed [1,11,12,13,14,15,16,17,18]. Alike the probiotic bacteria, several beneficial fungi and/or their bioactive compounds are applied to control diseases. These fungal species are isolated either randomly or systematically [19,20,21,22,23,24,25]. Amongst commercialized fungi, Aspergillus oryzae, Coniothyrium minitans, Phlebiopsis gigantea and Trichoderma (teleomorph Hypocrea ) spp., are able to suppress diseases and promote the growth of various hosts, mainly terrestrial crops and some animals such as cattle [27,28,29,30]. For fish, Trichoderma viride enhanced body weight and reduced mortality of Nile tilapia exposed to Saprolegnia sp. . The commercial product HetroNex, containing Trichoderma viride and Trichoderma harzianum, has been developed to control fungal and oomycete diseases caused by Fusarium, Lagenidium and Saprolegnia in aquaculture ponds of fish, prawn and shrimp .
In a fungal diversity study of the marine sponge Dragmacidon reticulatum, Trichoderma represented one of the most abundant genera among the isolated fungi . To date, however, still little is known about the fungal community in aquaculture or aquatic environments [18,34]. Previously, we showed by clone library analyses that the oomycete community associated with Saprolegnia-infected (diseased) and healthy salmon eggs from a commercial fish hatchery were dominated by Saprolegnia with no difference in the number and pathogenicity of the Saprolegnia isolates present in either diseased or healthy salmon egg batches . Based on terminal restriction fragment length polymorphism (T-RFLP) analysis and clone library sequencing, also no obvious differences were observed in the fungal community composition between the diseased and healthy salmon egg batches . The clone library consisted of 209 fungal clones, the majority of which belonged to the Ascomycota. More specifically, 139 clones were classified as Microdochium (teleomorph Monographella) [18,35,36]. To elucidate the role of fungi in the protection of salmon eggs against Saprolegniosis, we isolated and (phylogenetically) characterized fungi from diseased and healthy salmon eggs. Their abundance in diseased and healthy salmon egg batches and their activity against Saprolegnia diclina were investigated here.
2. Results and Discussion
2.1. Isolation of Fungi from Diseased and Healthy Salmon Eggs
Previously, Saprolegnia-infected (diseased) and healthy salmon eggs and their corresponding incubation water were sampled from a commercial fish farm (N = 6 for diseased eggs and N = 6 for healthy eggs) . Per sample, one or two salmon eggs were placed on potato dextrose agar to allow fungal outgrowth (Figure 1). We obtained and purified 20 fungal isolates in total and by ITS sequencing identified three different genera, Microdochium, Trichoderma (both Ascomycota) and Mortierella (Zygomycota). Microdochium and Mortierella were the most represented genera in our previous clone library sequence analysis. Interestingly, Trichoderma was not detected in the previous analysis, probably due to the limited number of sequenced clones, the specificity of the primers or the efficiency of the PCR reaction . The two Mortierella isolates (BLAST identity of 100% in Genbank database) were isolated from only one healthy salmon egg sample. Here we aimed at comparing isolates obtained from multiple replicate samples of diseased and healthy eggs. Therefore, we focused on the Ascomycota isolates for the subsequent analyses described below.
Microdochium species are known as snow molds, and some are pathogenic to plants [37,38,39,40,41,42,43,44]. Microdochium nivale and Microdochium majus are two of the main causative agents of Fusarium head blight , whereas Microdochium lycopodinum and Microdochium phragmitis were isolated from plants without causing disease [45,46]. M. phragmitis was endophytic in common reed and was more present in flooded habitats than the closely related Microdochium bolleyi . Some Microdochium species were antagonistic to the plant pathogen Verticillium dahliae . Amongst our nine Microdochium isolates, five were isolated from diseased and four from healthy salmon eggs (Table 1). The origin of our Microdochium isolates was possibly from the catchment area, which was the water source for the salmon egg incubators . Based on the phylogenetic analyses of internal transcribed spacer (ITS) sequences, all the nine Microdochium isolates are closely related to M. lycopodinum and M. phragmitis, and no distinct separation is observed between isolates from diseased or healthy salmon eggs (Figure 2). Quantitative PCR using M. lycopodinum/M. phragmitis specific primers showed and confirmed that M. lycopodinum/M. phragmitis was detected in equal amounts in total DNA samples obtained from diseased and healthy salmon egg samples (Figure 3). One Microdochium isolate (749F1) inhibited the hyphal growth of Saprolegnia diclina on 1/5th strength potato dextrose agar (1/5PDA) (Table 1, Figure 4a) suggesting the secretion of enzymes or other bioactive metabolites.
|Genus||Strain No.||Salmon Egg Sample||Activity of Culture Filtrate||Dual Culture Assay on 1/5PDA||Hyphal Interaction with S. diclina Microscopically|
|Microdochium||41F2||Diseased||Not inhibitory||Not inhibitory||Not observed|
|684F5||Diseased||Not inhibitory||Not inhibitory||Not observed|
|736F1a||Diseased||Not inhibitory||Not inhibitory||Not observed|
|736F1b||Diseased||Not inhibitory||Not inhibitory||Not observed|
|1056F2||Diseased||Not inhibitory||Not inhibitory||Not observed|
|765F1a||Healthy||Not inhibitory||Not inhibitory||Not observed|
|765F1b||Healthy||Not inhibitory||Not inhibitory||Not observed|
|749F1||Healthy||Not inhibitory||Inhibitory||Not observed|
|749F2||Healthy||Not inhibitory||Not inhibitory||Not observed|
|Trichoderma||684F1||Diseased||Not inhibitory||Not inhibitory||Coiling, papilla-like structure|
|1056F1||Diseased||Not inhibitory||Not inhibitory||Papilla-like structure|
|1152F1||Diseased||Not inhibitory||Not inhibitory||Inconclusive|
|762F1a||Healthy||Not inhibitory||Not inhibitory||Coiling|
|762F1b||Healthy||Not inhibitory||Not inhibitory||Coiling|
|762F2||Healthy||Not inhibitory||Not inhibitory||Papilla-like structure|
|764F1||Healthy||Inhibitory||Not inhibitory||Papilla-like structure|
|764F2||Healthy||Not inhibitory||Not inhibitory||Coiling, papilla-like structure|
|764F3||Healthy||Not inhibitory||Not inhibitory||Coiling, papilla-like structure|
To date, not much is known about Microdochium in aquatic environments, aquaculture or aquatic animals . Also not much is known about the bioactive compounds produced by Microdochium. Bhosale et al. (2011) reported that the active compound cyclosporine A, extracted from an estuarine M. nivale, has the potential to be applied pharmaceutically to control diseases caused by some dermatophytes and Aspergillus species in human and animals ; Santiago et al. (2012) reported that the extract of M. phragmitis, which was isolated from Antarctic angiosperms, showed cytotoxic activity against a human tumoral cell line . Therefore, further experiments are needed to decipher the bioactive potential capacity of our Microdochium isolates, especially their interaction with pathogens from cold water environments, like Saprolegnia spp.
Most Trichoderma species are applied in agriculture as biocontrol agents against various plant-associated bacterial, fungal and oomycete pathogens, such as Clavibacter, Fusarium and Phytophthora [26,51,52]. Trichoderma species are capable of producing a range of extracellular compounds to suppress plant pathogens, such as enzymes, fungicidal compounds and antibiotics; they can also promote plant growth via symbiotic association with plant hosts [26,51,53,54,55]. Trichoderma is commonly isolated from terrestrial environments, such as soil and wood , but also from aquatic environments like freshwater (drinking water) and marine water [33,56,57,58,59,60]. Marine Trichoderma atroviride and Trichoderma asperelloides suppressed disease caused by Rhizoctonia solani on beans and enhanced defence responses against pathogenic Pseudomonas syringae pv. Lachrimans on cucumber seedlings . Some other marine-derived Trichoderma strains were capable of producing antagonistic compounds against cancer, diabetes, cancer cell lines or pathogenic Staphylococcus epidermidis; such compounds include tandyukisins from Trichoderma harzianum OUPS-111D-4, pyridones from Trichoderma sp. MF106, and trichoketides from Trichoderma sp. TPU1237 [58,61,62].
It was suggested that Trichoderma may have the potential to also control infectious diseases in aquaculture . Among our nine Trichoderma isolates, three were isolated from diseased and six from healthy salmon eggs (Table 1). Quantitative PCR with Trichoderma-specific primers showed that Trichoderma was present in higher abundance in total DNA samples of healthy than of diseased salmon eggs (Figure 3), although some variation in results were observed between replicated PCR reactions on the same DNA samples (Supplementary Material, Figure S1). The total fungal community did not differ in abundance between healthy and diseased salmon eggs (Figure 3). Collectively, these results suggest that Trichoderma is more enriched in healthy salmon egg samples than in diseased salmon egg samples. Phylogenetic analyses based on ITS sequences showed that all nine Trichoderma isolates belonged to the Trichoderma section  and no apparent separation was observed between isolates from diseased or healthy salmon eggs based on ITS sequences (Figure 5). However, phylogeny based on sequences of the translation elongation factor 1 alpha (tef1) clearly separated the Trichoderma isolates from diseased and healthy salmon eggs (Figure 6). Our nine Trichoderma isolates and the Trichoderma viride reference strains [65,66] formed three clades of Trichoderma viride. These results suggest that next to a quantitative difference also a qualitative difference in Trichoderma populations from diseased and healthy salmon eggs.
In terms of extracellular activity, we observed that the culture filtrate of only one Trichoderma isolate showed inhibition of hyphal growth of S. diclina (Table 1). Dual culture assays did not show inhibition of hyphal growth of S. diclina by any of the Trichoderma isolates tested (Table 1, Figure 4a). However, the hyphae of most Trichoderma isolates coiled around or produced a papilla-like structure on the hyphae of S. diclina (Table 1 and Figure 4b) . The coiling and papilla-like structures suggest attachment of Trichoderma hyphae to S. diclina hyphae. Coiling is required for mycoparasitism but not all coiling leads to mycoparasitism [26,54,55,67]. The formation of papilla-like structures in the interaction with S. diclina could indicate the start of mycoparasitic invasion by Trichoderma; these structures have been shown to induce hyphal breakdown of various hosts [26,54,55,68,69].
Even though Trichoderma species are commonly considered beneficial fungi, some Trichoderma strains, including T. harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma pseudokoningii and Trichoderma viride, maybe pathogenic to human [72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87]. Some marine Trichoderma were associated to contaminated mussels and some were even toxic to aquatic animals, such as Artemia larvae [25,88]. Therefore, evaluations of the adverse effects of our Trichoderma isolates on the environment and humans are needed.
Previous work by Abdelhamid et al. (2007) indicated that T. viride can enhance body weight and reduce mortality of Nile tilapia treated with Saprolegnia sp. . Our isolates belong to the T. viride clade and are, to our knowledge, the first characterized Trichoderma from salmon eggs. Collectively, our results pointed to both quantitative and qualitative differences in Trichoderma population between diseased and healthy salmon eggs. These analyses suggest a potential role of Trichoderma species in the protection of salmon eggs from S. diclina. Hence, our Trichoderma isolates and/or their metabolites, especially isolate 764F1 and its bioactive compounds, may have the potential to be applied in aquaculture. To this end, in vivo experiments should be conducted to determine the beneficial effects of our Trichoderma isolates in controlling Saprolegniosis and other aquaculture diseases.
3. Experimental Section
3.1. Phylogenetic Analysis of Microdochium and Trichoderma Isolates
Fungal isolation from salmon eggs was described previously by Liu et al. . DNA isolation, internal transcribed spacer (ITS) rRNA sequencing and phylogenetic analyses of fungal isolates were conducted as described by Liu et al. . For Trichoderma isolates, additional phylogenetic analysis was conducted with translation elongation factor 1 alpha (tef1) sequences. The tef1 gene was amplified by primer set EF1-728F  and TEF1LLErev . The evolutionary distances of the phylogenetic trees were computed using the Kimura 2-parameter method  for Microdochium ITS sequences, Tamura 3-parameter method  for Trichoderma ITS sequences and Tamura-Nei method  for tef1 sequences.
3.2. Culture Filtrate Activity of Microdochium and Trichoderma Isolates
One agar plug of each Microdochium and Trichoderma isolate was pregrown in 6 mL 1/5th strength potato dextrose broth (1/5PDB, Difco™, Franklin Lakes, NJ, USA) for one week at 20–25 °C. Each culture was lyophilized, the pellet dissolved in 6 mL fresh 1/5PDB and filter-sterilized through a 0.2 μm filter (Whatman™, Freiburg, Germany). 1 mL culture filtrate solution was added into a well of 24 well suspension culture plate (Greiner bio-one, Cellstar®, Frickenhausen, Germany) and one agar plug of Saprolegnia diclina 1152F4 was added into each well. After incubation at 14–15 °C for four days the effect of the culture filtrates on hyphal growth of S. diclina was determined.
3.3. Dual Culture Assay
One agar plug of each of the Microdochium or Trichoderma isolates and one agar plug of S. diclina were placed at two opposite sides of 1/5th strength potato dextrose agar (1/5PDA). After incubation for 6 days at 20–25 °C, inhibition of hyphal growth of S. diclina was determined and plates were stored for one to two months at 4 °C until microscopic analyses were performed. Hyphal interactions were observed under a Nikon 90i epifluorescence microscope (Nikon Instruments Europe BV, Amsterdam, The Netherlands) with brightfield settings and accomplished with Nikon NIS-elements.
3.4. Quantification of Total Fungi, Microdochium and Trichoderma in Salmon Egg Incubation Water
DNA extraction from salmon egg samples and storage was described in Liu et al. . All DNA samples were thawed on ice and normalized to 5 ± 1 ng·μL−1. To quantify total fungi in the water samples, ITS rRNA genes were amplified with ITS4  and ITS9  primers in 12 μL volumes, each consisted of 5 μL of DNA template, 5.8 μL BIOLINE 2x SensiFAST SYBR No-ROX mix, 0.1 μL ITS4 primer (10 mM), 0.1 μL ITS9 primer (10 mM) and 1 μL BSA (0.1 mg·mL−1). The quantitative PCR program consisted of 1 cycle at 95 °C for 5 min, 45 cycles at 95 °C for 20 s, 55 °C for 20 s, 72 °C for 30 s, 82 °C for 15 s. To quantify Microdochium lycopodinum/Microdochium phragmitis in the water samples, MPF (5′-AAGGTACCCGAAAGGGTGCTGG-3′) and MPR (5′-GAATTACTGCGCTCAGAGTACGT-3′) primers were designed and firstly the accuracy was verified by PCR using the genomic DNA isolated from M. phragmitis CBS 285.71 and Microdochium nivale var. nivale CBS 110.94 as template (Supplementary Material, Figure S2). The quantitative PCR program consisted of 1 cycle at 95 °C for 5 min, 45 cycles at 95 °C for 20 s, 60 °C for 20 s, 72 °C for 30 s, 82 °C for 15 s. To quantify Trichoderma in the water samples, Trichoderma specific genes were amplified with ITS1TrF and ITS4TrR primers  and QIAGEN Rotor-Gene® SYBR® Green PCR Master Mix 2x was used. The quantitative PCR program consisted of 1 cycle at 95 °C for 5 min, 45 cycles at 95 °C for 10 s, 51 °C for 10 s, 72 °C for 20 s, 82 °C for 10 s. Genomic DNA of Trichoderma isolates 1152F1 and 762F1b, and Microdochium isolates 736F1a and 749F1 was isolated with PowerSoil® DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. Dilution series of DNA of each strain was prepared at 5, 5 × 10−1, 5 × 10−2, 5 × 10−3, 5 × 10−4, 5 × 10−5, 5 × 10−6, 5 × 10−7 ng·μL−1 and used as standards (Supplementary Material, Figure S3).
3.5. Nucleotide Sequence Accession Numbers
All DNA sequences have been deposited in GenBank. The accession numbers for the internal transcribed spacer sequences of Trichoderma, Microdochium and Mortierella are KU202214-22, KU202223-31 and KU202232-33, respectively. The accession numbers for the sequences of translation elongation factor 1 alpha of Trichoderma isolates are KU202234-42.
Aquaculture has become one of the fastest developing animal food sectors , partly due to regulations to protect wild fish populations from overfishing and the increased demand for fish products . To support this increase in food demand, aquaculture production is gradually intensifying, but effective and sustainable strategies are needed to suppress emerging diseases including Saprolegniosis. Very few studies have demonstrated the beneficial activity of fungi against aquatic pathogens [31,95]. Our study is, to our knowledge, the first to establish correlations between the frequency/occurrence of indigenous fungal communities (Trichoderma and Microdochium species) and the health status of salmon eggs in a commercial hatchery. Our study is also the first to assess the diversity among Trichoderma and Microdochium isolates from aquaculture samples. The traditional plate assays provided informative results showing the potential antagonistic activity of our Trichoderma isolates obtained from salmon eggs against the pathogen Saprolegnia diclina. These results demonstrated the basic characters of our Trichoderma isolates, which provide a good starting point for future analyses on the molecular basis of Trichoderma-Saprolegnia interactions. Further in vitro and in vivo tests are needed to confirm their beneficial protective activity in situ. The role of Trichoderma in Saprolegnia disease suppression is especially interesting, since Trichoderma was shown to be more abundant in healthy salmon eggs than in diseased ones and showed a mycoparasitic interaction with Saprolegnia. Our study provides a framework to isolate and monitor putative protective fungi in Saprolegnia control and possibly other emerging diseases in aquaculture.
Supplementary materials can be found at https://www.mdpi.com/1422-0067/17/1/140/s1.
This work was financially supported by SAPRO (Sustainable Approaches to Reduce Oomycete (Saprolegnia) Infections in Aquaculture, 238550), a Marie Curie Initial Training Network funded by the European Commission under Framework Program 7; ParaFishControl (Advanced Tools and Research Strategies for Parasite Control in European farmed fish, 634429), a Research and Innovation action funded by the European Commission under HORIZON 2020. The funds for covering the costs to publish in open access are received from ParaFishControl. We thank Klaas Bouwmeester (Wageningen University) for his help with microscopic work. We thank Emilia Hannula, Rosalinde M. Keijzer, Saskia Gerards, Agata Pijl (NIOO-KNAW) and Johnny Soares (Agronomic Institute—IAC, Brazil) for their advices in quantitative PCR. This manuscript is publication number 6010 of Netherlands Institute of Ecology (NIOO-KNAW).
Conceived and designed the experiments: Yiying Liu, Christin Zachow, Irene de Bruijn and Jos M. Raaijmakers. Performed the experiments: Yiying Liu. Analyzed the data: Yiying Liu, Christin Zachow, Irene de Bruijn. Created figures: Yiying Liu, Irene de Bruijn. Wrote the paper: Yiying Liu, Irene de Bruijn, Jos M. Raaijmakers. Contributed to phylogenetic analyses, microscopic observation and quantitative PCR design: Christin Zachow. Designed M. lycopodinum/M. phragmitis specific primers: Yiying Liu, Irene de Bruijn. Contributed to review of the manuscript: all authors.
Conflicts of Interest
The authors declare no conflict of interest.
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