A novel fungus Penicillium canescens LS-4.2 with algicidal activity against the toxic cyanobacterium Microcystis aeruginosa

. In this study, the fungus strain LS-4.2 isolated from the bottom sediments of Lake Lower Suzdalskoe was tested for its algicidal activity. We identified the strain LS-4.2 as Penicillium canescens summing its morphological characteristics with the reported DNA sequence. We revealed that the filtrate of a 7-day culture of the fungus suppressed the cell growth of toxic Microcystis aeruginosa . Our results showed that the filtrate caused rapid M. aeruginosa growth inhibition up to complete cell lysis recorded after 4 days. Living fungal mycelia did not suppress the growth of cyanobacterium. According to the results of this study we suppose that strain LS-4.2 may be a potential bioagent in the control of cyanobacterial blooms.


Introduction
Harmful cyanobacterial blooms (HCBs) are currently being recorded in freshwater worldwide [1].Microcystis aeruginosa is among the most common species of the blooming cyanobacteria, it should be noted that many strains of the genus Microcystis are able to form toxic metabolitesmicrocystins [2].It was also reported about the cyanobacterial bloom from water bodies located in the St. Petersburg region, including Lake Lower Suzdalskoe (Suzdal).The surface area of Suzdal is 97 ha and the mean depth is 3.0 m.The citizens of St. Petersburg region use the water of this lake for recreational purposes.
The cyanobacteria Microcystis spp.and Planktothrix agardhii, a number of strains of which are known to produce cyanotoxins, dominate the lake Suzdal water for most of the summer season.In eutrophic and shallow Suzdal nine microcystins were found [3].Microcystins (MCs) are cyclic heptapeptides that inhibit enzyme activity in hepatocytes.High doses of MСs cause extensive hemorrhages in the liver, and long-term exposure to low doses of MCs leads to malignant formations [4][5].
Many approaches have been proposed to combat HCBs, including physical and chemical methods [6][7].However, most of the physical and chemical methods are expensive, and potentially hazardous to the environment.Biological methods are considered the most economical and environmentally friendly for inhibiting cyanobacteria.Many aquatic organisms have been studied for their ability to limit the growth of cyanobacteria.Viruses, bacteria, fungi, actinomycetes, protozoa, zooplankton organisms, mollusks have been investigated as potential agents of biological control in various studies.The activity of potential biocontrol agents varies from highly specific parasitism and predation to nonspecific forms of action, such as filtration, and competition for resources and the release of metabolites that inhibit cyanobacterial growth [2].
Many of these methods involve the use of microorganisms with anticyanobacterial activity [8].Most of the known anticyanobacterial microorganisms are bacteria, belonging mainly to Proteobacteria, Actinomycetes, Bacteroidetes, Firmicutes and Thermus [2,9].
However, the degradation of cyanobacteria cells by fungal strains is currently insufficiently explored.Only 15 fungal strains belonging to genera Ascomycetes (9 strains) and Basidiomycetes (6 strains) have been found to have the anticyanobacterial effects [2,10].
In this study, we investigated the ability of novel fungus Penicillium canescens LS-4.2 to suppress the growth of the toxic cyanobacterium Microcystis aeruginosa.

Fungal isolation, identification and cultivation.
The fungal strain LS-4.2 was isolated from bottom sediment collected from eutrophic Lake Lower Suzdalskoe on a Czapek's Agar with streptomycin (100 U mL −1 ) as selective agent [11].
The fungal strain LS-4.2 was identified on the strength of morphological, cultural characteristics using determinant [12] and on ITS1-5.8S-ITS2DNA region sequencing, which was amplified using universal primers pairs: ITS1 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4 5'-TCCTCCGCTTATTGATATGC-3' [13].Isolation, amplification and sequencing of fungal genomic DNA were performed according to the previously described procedure [11].The nucleotide sequence of the PCR products was determined using an ABI 3500xl genetic analyzer (Applied Biosystems, USA) at the Russian Collection of Agricultural Microorganisms (RCAM, WDCM 966).The sequences were compared with related sequences available in the GenBank databases using BLAST analysis (http://www.ncbi.nlm.nih.gov).
Fungus strain LS-4.2 was cultured in 250 mL Erlenmeyer flask with 50 mL of Czapek's liquid medium at 230 rpm (Certomat BS-1 rotary shaker) and 28±1°C.The fungal filtrate of a 7-day culture and living fungal mycelia were used for algicidal experiments.Algal culture.
M. aeruginosa strain CALU 973 was acquired from Resource Center "Culture Collection of Microorganisms" of the Saint-Petersburg State University.The cyanobacterium was cultured under static conditions in 250-mL Erlenmeyer flasks with the BG11 liquid medium (100 mL) at a temperature 25±1°C, an illumination level of 1000 lux, a light/dark conditions of 12 h/12 h.Сell density of the culture was defined by OD680 (Genesys 10 UV scanning spectrophotometer).
Chlorophyll a was extracted from the biomass with 90% acetone at 4°C for 24 h.Optical densities of the acetone extract of chlorophyll a at wavelengths of 664, 647, and 630 nm were determined on a Genesys 10UV scanning spectrophotometer.The concentration of chlorophyll a was calculated by formular (1) chlorophyll a (mg/l) = 11.85 × A664 ˗ 1.54 × A647 ˗ 0.08 × A630 , 02 where А664, А647, А630 -optical densities of the acetone extract of chlorophyll a at wavelengths of 664, 647, and 630 nm, respectively.Experimental designs.
An aliquot of algal culture was introduced into flasks with 100 mL BG11 medium to give an initial cell density OD680=0.05.The living fungal mycelia (100 mg dw) and the fungal filtrate (10% (v/v)) were added into the flasks with algae.Algal culture with added BG11 medium (10% (v/v) for volume equalization and algal culture with Czapek's medium (10% (v/v) served as сontrol 1 and сontrol 2 respectively.All variants were cultivated under conditions for M. aeruginosa.The cyanobacterial growth was estimated daily.Three replicates were performed for treatment and сontrols.

Statistical analysis.
Statistical analysis and graphical presentation of the results were carried out using Microsoft Excel 2007 and Past 4.0 software.The statistical significance of differences between the variants was determined with one-way ANOVA (Mann-Whitney U-test (p<0.05).Data were presented as the arithmetic mean ± standard deviation (SD) of three independent biological replicates.

Results
The isolated strain LS-4.2 on solid nutrient Czapek-Dox medium on the 7th day of growth forms colonies consisting of a dense felt plexus of aerial mycelium, radially striated, with abundant conidial sporulation from grayish-greenish to smoky gray.Colonies on the 14th day at a temperature of 25°C reach a diameter of 21-25 cm.Reverse colonies is goldenyellow, than dark brown.ITS similarities between the strain LS-4.2 and other strains within genus Penicillium -Penicillium arizonense strain IBT: 122989T, Penicillium canescens NRRL910T, Penicillium radiatolobatum CBS 340.79T were 99.81% at 99-100% query cover.Strain LS-4.2 had the same ITS sequences as the type strains of P. arizonense, P. canescens and P. radiatolobatum.Though, based on macromorphological analysis, this strain is more likely to belong to the species P. canescens.Thus, based on the analysis of morphological characteristics and ITS sequencing, strain LS-4.2 was tentatively identified as Penicillium canescens Sopp.
In our study the LS-4.2 fungal filtrate of the 7-day culture greatly suppressed the growth cells of M. aeruginosa (Figures 1 and 2).We observed that inhibitory effect was time-dependent (Figure 2).The insignificant (by 17%), but statistically significant level of M. aeruginosa cells lysis was observed already after the first day of incubation of cyanobacteria with fungal filtrate cells was observed.The cyanobacterial cells were almost completely lysed after 4 days.These results are consistent with the data on changes in the content of chlorophyll a in the medium (Figure 3).The living fungal mycelia did not suppress the growth of cyanobacterium (data not shown).

Discussion
Many fungi of the genus Penicillium are destructors of toxic anthropogenic and natural environmental pollutants.It is known about the ability of the strain Penicillium canescens to decompose toxic polycyclic aromatic hydrocarbons [14].Meanwhile, as far as with known, there is no information on the antialgal activity of Penicillium canescens.
In the present study, for the first time, the anticyanobacterial activity of Penicillium canescens was reduced.The fungal filtrate of strain LS-4.2 inhibited the cell growth of M. aeruginosa and chlorophyll content in the medium.Our strain P. canescens LS-4.2 removed 100% of cyanobacterial cells within 4 days.This data suggest that strain LS-4.2 secrets antialgal substances into culture medium.
Most antialgal fungi have been found to have an indirect mechanism of action, releasing algicidal compounds that inhibit the growth of cyanobacteria [16,18,19].An indirect mechanism of algicidal activity was found in the fungus Trichoderma citrinoviride against M. aeruginosa.The addition of the culture liquid filtrate of the ascomycete fungus T. citrinoviride to the medium with M. aeruginosa cells inhibited the growth of cyanobacteria after 48 h of incubation [19].It is noted that the algicidal activity of this ascomycete exceeds the activity of some basidiomycetes.
Unlike ascomycetes, basidiomycetes exhibited algicidal activity upon direct contact with cyanobacterial cells [2,10].The species of fungi Irpex lacteus T2b, Trametes versicolor F21a and Bjerkandera adusta T1 with an initial dry weight of inoculum of 142, 97.6 and 78.4 mg, respectively, significantly inhibited the growth of Microcystis aeruginosa (initial concentration of chlorophyll a 600-700 µg/l) on average after 60 hours.The T. versicolor F21a strain showed the highest algicidal activity, complete inhibition of algae was observed after 30 hours of incubation.It was noted that neither inactivated fungal cells nor culture filtrates inhibited the growth of cyanobacteria, indicating that the algicidal activity of these fungi was due to a direct effect on cyanobacteria cells [20].

Conclusion
In summary an antialgal fungal strain identified as Penicillium canescens LS-4.2 was isolated from the bottom sediment from Lower Lake Suzdalskoe.The obtained results show that extracellular substances released from P. canescens LS-4.2 into medium lysed the toxic cyanobacterium M. aeruginosa cells.The fungus P. canescens could be used as cyanobacterial blooms control agent.