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Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species
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Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species Luciano BOSSOa,*, Rosalia SCELZAa, Rosaria VARLESEa, Giuseppe MECAb, Antonino TESTAa, Maria A. RAOa, Gennaro CRISTINZIOa n. 100, 80055 Portici (Naples), Italy Department of Agriculture, University of Naples Federico II, via Universita Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjassot, Spain
a
b
article info
abstract
Article history:
Bioremediation and biological-control by fungi have made tremendous strides in numer-
Received 8 May 2015
ous biotechnology applications. The aim of this study was to test Byssochlamys nivea and
Received in revised form
Scopulariopsis brumptii in sensitivity and degradation to pentachlorophenol (PCP) and in bi-
5 January 2016
ological-control of Phytophthora cinnamomi and Phytophthora cambivora. B. nivea and S.
Accepted 6 January 2016
brumptii were tested in PCP sensitivity and degradation in microbiological media while
Corresponding Editor:
the experiments of biological-control were carried out in microbiological media and soil.
Nabla Kennedy
The fungal strains showed low PCP sensitivity at 12.5 and 25 mg PCP L1 although the hyphal size, fungal mat, patulin, and spore production decreased with increasing PCP concen-
Keywords:
trations. B. nivea and S. brumptii depleted completely 12.5 and 25 mg PCP L1 in liquid
Bioremediation
culture after 28 d of incubation at 28 C. Electrolyte leakage assays showed that both fungi
Electrolyte Leakage Assay
have low sensitivity to 25 mg PCP L1 and produced no toxic compounds for the plant. B.
Fungi
nivea and S. brumptii were able to inhibit the growth of the two plant pathogens in labora-
Oomycetes
tory studies and reduce the mortality of chestnut plants caused by two Phytophthorae in
Patulin
greenhouse experiments. The two fungal strains did not produce volatile organic com-
Plant pathogens
pounds able to reduce the growth of two plant pathogens tested. ª 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Bioremediation is defined as the process whereby living organisms are able to transform environmental contaminants into less toxic forms. It uses naturally occurring bacteria, fungi or plants to degrade or detoxify substances hazardous
to human health and/or the environment (Crawford & Crawford 1997; Vidali 2001; Olguin 2003; Petroselli et al. 2014). Biological control (biocontrol) refers to the purposeful utilization of introduced or resident living organisms to suppress the activities and populations of one or more plant
* Corresponding author. Tel.: þ39 081 2539371, þ39 3290758021(mobile). E-mail address: [email protected] (L. Bosso). http://dx.doi.org/10.1016/j.funbio.2016.01.004 1878-6146/ª 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
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pathogens to improve plant health. Disease suppression by biocontrol agents is the sustained manifestation of interactions among the plant, the pathogen, the antagonist, the microbial community on and around the plant, and the physical environment (Handelsman & Stab 1996; Howell 2003; Lorito et al. 2010). The use of beneficial fungi and bacteria for bioremediation and biocontrol of plant pathogens has made tremendous strides in numerous biotechnology applications. In recent years the need to find a global approach to environmental and agricultural issues has set the challenge to discover microorganisms which are useful for both bioremediation and biocontrol research (Griffin 2014). This approach is primarily focused on how to use microorganism strains to inhibit the dangerous advance of plant pathogens and to deplete environmental contaminants (Sylvia et al. 2005; Singh et al. 2011). The fungi that play important roles in biocontrol and bioremediation strategies are numerous; some of these are grouped as mycorrhizal and endophytic fungi (Griffin 2014). The endophytic fungus Phomopsis sp. has been found to use the 4-hydroxybenzoic acid as the only carbon source (Chen et al. 2011) and to inhibit the growth of Physocnemum brevilineum which is a vector of Ceratocystis ulmi, an important elm pathogen (Webber 1981). The fungi belonging to genus Glomus are arbuscular mycorrhizas used in the biocontrol n-Aguilar & Barea 1996) of soil-borne plant pathogens (Azco and as heavy metal biosorbents in soil (Leyval et al. 1997). Filamentous fungal species belonging to the genus Trichoderma are able to counteract some plant pathogens by means of mycoparasitism and antibiosis (Howell 2003: Lorito et al. 2010) and simultaneously to deplete pollutants including heavy metals, pentachlorophenols (Bosso & Cristinzio 2014) and polycyclic aromatic hydrocarbons (PAHs) (Tripathi et al. 2013). Aspergillus flavus was used as a biosorbent of heavy metals (Deepa et al. 2006), phenol degrader (Ghanem et al. 2009; Bosso & Cristinzio 2014) and promoter of Phytophthorae’s growth inhibition (Evidente et al. 2009). Penicillium spp. has demonstrated an excellent ability to degrade different xenobiotic compounds such as phenolic compounds, ~ o 2009). Nevertheless some PAHs and heavy metals (Leita strains of genus Penicillium were also used in biocontrol. In fact, Penicillium funiculosum and Penicillium janthinellum have been able to limit the Phytophthorae root rots of azalea (Ownley & Benson 1992; Fang & Tsao 1995). Some species of genus Verticillium have been found to remove petroleum products and PAHs in soil (Gadd 2001; Singh et al. 2011) and to control numerous plant pathogens such as fungi, bacteria rillon & Ramawat 2012). Other interestand nematodes (Me ing ecological groups are for example the white-rot fungi. Trametes versicolor has shown promising biocontrol activities ~ as & Martınez 1996) against Fusarium oxysporum (Ruiz-Duen and represents one of the most important organisms used in bioremediation actions (Gadd 2001; Singh 2006; Bosso & Cristinzio 2014). Pentachlorophenol (PCP) is a toxic compound which is widely used as a wood treatment agent and general biocide. It is persistent in the environment and has been classified as a priority contaminant to be reclaimed in many countries. In fact, uncontrolled PCP uses and releases have
L. Bosso et al.
caused contamination of soil, water and ground water. Although PCP is recalcitrant to biodegradation, numerous bacterial and fungal isolates have been reported to be able to degrade it (Gadd 2001; Singh 2006; Bosso & Cristinzio 2014). In the present study, two fungal strains which were isolated from artificially PCP-contaminated soil during a longterm bioremediation study and identified as Byssochlamys nivea (Westling 1909) and Scopulariopsis brumptii (SalvanetDuval 1935) (Scelza et al. 2008; Bosso et al. 2011; Bosso et al. 2015b), were tested in laboratory and greenhouse experiments to reach the following goals: 1 to evaluate their sensitivity to PCP and their capacity for contaminant degradation; 2 to assess their antagonistic effect vs Phytophthora cinnamomi (Rands 1922) and Phytophthora cambivora (Buisman 1927), soil-borne pathogens causing disease on many woody hosts (root and collar rot of adult trees and of seedlings in nurseries, plantations and forests) especially on plants belonging the genus Castanea (Vannini & Vettraino 2001).
Materials and methods Fungal strains and cultivation conditions Byssochlamys nivea and Scopulariopsis brumptii were stored into slant tubes containing potato dextrose agar (PDA; 5 g L1 potato; 20 g L1 dextrose; 15 g L1 agar) at 20 C at the laboratories of Forest Pathology of the Department of Agriculture (University of Naples Federico II, Italy).
Chemicals PCP (>99 % purity) was purchased from SigmaeAldrich (USA). All solvents and chemicals reagents were purchased from Carlo Erba Reagents (Italy).
Sensitivity test to PCP in plate culture Fungal sensitivity to PCP was evaluated in Petri dishes. The fungal strains were grown onto PDA at different PCP concentrations (12.5 and 25 mg L1) prepared previously by dissolving the suitable amounts of PCP in 5 ml of methanol and then in PDA (final volume 1 L). This step was necessary because the PCP is weakly insoluble in water. The controls were cultured on PDA having 5 ml L1 of methanol but without PCP. All samples were incubated at 25 C for 7 d. Sensitivity to PCP was determined by measuring the diameter (Ø) of the colony, daily mycelium growth rate (Tomasini et al. 2001), hyphal size (this was achieved measuring with an ocular micrometer at total magnification of 100 the hyphal thickness in mm) and spore production (St-Arnaud et al. 1996). A number of 100 measurements were carried out for each replicate to determine hyphal thickness and spore production.
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
B. nivea and S. brumptii like PCP degraders and fungal antagonists
Sensitivity test to PCP and PCP removal in liquid culture Fungal sensitivity to PCP was determined also in liquid culture. Two plugs of 8 mm Ø of active mycelia were cut from PDA cultures of 7 d old and were grown in potato dextrose broth (PDB; 5 g L1 potato; 20 g L1 dextrose) with 12.5 or 25 mg PCP L1, prepared as described above. The controls were cultured without PCP. Cultures were grown in 250 ml Erlenmeyer flask containing 100 ml of medium. All samples were incubated under shaking conditions at 120 rpm for 28 d at 28 C. Sensitivity test was evaluated measuring the dry weight fungal mat. The fungal mat was separated from PDB by filtration using Whatman Filter MN 640 d e 110 mm Ø (Macherey-Nagel). The fungal mat was washed with distilled water, dried in an oven at 50 C for 24 h and weighed. PCP concentration in the fungal mat and liquid medium was determined by HPLC in according to the methods described in Tomasini et al. (2001). Briefly, the samples were filtered and the biomass was put on a sonic bath for 10 min using a carbonate buffer, pH 11. These fractions and similarly the culture broth samples were filtered through a 0.22 mm Whatman membrane (Macherey-Nagel). PCP from each fraction, biomass and culture broth, was quantified by HPLC and these two values were as added to the total residual PCP. Samples were analysed by HPLC with a C-18 reverse column (mbondapack Waters) packed with R-Sil C-18 (18 mm). Mobile phase was 1 % acetic acid water solution and 1 % acetic acid acetonitrile solution (75:25) at a flow rate of 0.8 ml min1. Elution was monitored at 238 nm and quantified with an integrator (Waters).
Fungal growth in presence of PCP as sole carbon source The fungal strains were grown in liquid mineral medium enriched with PCP as the sole carbon source. Each fungus was inoculated into a flask containing 12.5 or 25 mg PCP L1 in a medium having the following composition (g L1): KH2PO4 (2.0); MgSO4$7H2O (0.5); CaCl2 (0.1); NH4Cl (0.12); ZnSO4$7H2O (0.1); MnCl2$4H2O (0.3). The control was obtained with the same mineral medium but having 10 g L1 of glucose as the carbon source. The medium pH was adjusted to 5.5 before sterilization. The flasks were inoculated with two plugs of 8 mm Ø of active mycelia taken from 7-day-old PDA cultures. The incubation was carried out on orbital shaker incubator at 120 rpm in the dark for 28 d at 28 C. After that, the dry weight of the fungal mat was measured as described above. The analytical methods used to determine the PCP concentration in the fungal mat and in the liquid mineral medium were extensively described in the previous paragraph.
Production of patulin in liquid culture enriched by PCP For patulin extraction, the method of Raiola et al. (2012) was applied. In particular, 10 ml of liquid culture of Byssochlamys nivea or Scopulariopsis brumptii, obtained after 28 d of incubation in PDB, were transferred into a 50 ml-plastic tube containing 15 g of Na2SO4, 2 g of NaHCO3 and 10 ml of extraction solution (ethyl acetate/hexane 60:40, v/v) were added. The tube was shaken for 3.5 min on a mechanical
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shaker (Inter-Continental equipment, Hidalgo, TX) and then centrifuged at 2000 rpm for 1 min at 4 C. The supernatant (2.5 ml) was immediately placed onto an unconditioned Strata C18-E solid phase extraction column (Phenomenex, USA) which was then washed with 3 ml of the extraction solution adjusting the flow to one drop per second by using slight air pressure. The eluate was collected in a 10 ml-plastic test tube containing 50 mL of acetic acid. The solvent was evaporated at max 40 C for about 35 min in a vacuum centrifuge Thermo Savant (Speed Vacuum Thermo Electron Corporation Milford, MA, USA) and 1 ml of acidulated water (pH 4) was used to dissolve the sample. The solution was mixed with a vortex (Biosan MSV-3500, Lietsa, Finland) filtered with 0.22 mm filters (Phenomenex, Palo Alto, CA) and injected in the HPLC apparatus (Shimadzu-Japan) equipped with an autosampler SIL-20A, two pumps LC-20AD and a UV/vis detector SPD-20A set at 276 nm wavelength. The column was a Gemini 5 mm C18, 110 A (150 mm 2 mm) (Phenomenex, CA, USA). The mobile phase was water containing 1 % acetic acid (A) and methanol (B) (95/5, v/v) in isocratic conditions. The flow rate was of 1 ml min1. Under these chromatographic conditions, the retention time for patulin was 14.0 0.1 min.
Electrolyte Leakage Assay (ELA) Electrolyte Leakage Assay (ELA) is a test that assesses the toxicity in plant tissues, as this toxicity is linked to the damage on plant cell wall and membrane induced by toxic compounds (Peever & Higgins 1989). ELA was carried out on both fungi to assess: i) the toxic effect of PCP on mycelium and; ii) the potential damage on plant cell wall and membrane due to fungal metabolites. Regarding the first purpose, six plugs of 5 mm Ø of active mycelia were taken from 7-day-old water agar cultures (WA, Carlo Erba) and incubated in the dark for 24 h in beakers containing a water solution of 25 mg PCP L1. The control was prepared incubating the fungus with only distilled water. After the incubation all fungal plugs were washed and transferred into a beaker containing 15 ml of distilled water. The ELA measurements were conducted after 0.5, 1, 2, 4, 7, 8, and 24 h. For the second purpose, ten tomato (Solanum lycopersicum) stem pieces (5-mm long) were incubated overnight in a culture filtrate of Byssochlamys nivea or Scopulariopsis brumptii. The fungal culture filtrate was obtained from PDB medium with active mycelia cultured for 7 d. The culture filtrate was separated from the mycelium by filtration with Whatman Filter MN 640 d e 110 mm Ø (Macherey-Nagel). The control was obtained by incubating the stem in only PDB. After the incubation, all the stem pieces were washed and transferred into a beaker containing 15 ml of distilled water. The measurements were set hourly except for the first and last analyses which were made after 0.5 and 24 h, respectively. In all the experiments, the conductance was measured in microsiemens (mS) using a conductivity-meter with 20 electrodes (range 20e200 mS cm1; K ¼ 1). The conductance values were calculated as the difference from the reading at the beginning of the assay (increments in microsiemens from t0) (Evidente et al. 2009).
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
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Antagonistic assays of Byssochlamys nivea and Scopulariopsis brumptii versus Phytophthora cambivora and Phytophthora cinnamomi under laboratory and greenhouse conditions The antagonistic assays were carried out among our fungal strains against Phytophthora cambivora and Phytophthora cinnamomi under laboratory and greenhouse conditions. These two Chromisti are stored into PDA slant tubes at 18 C at the laboratories of Forest Pathology of the Department of Agriculture (University of Naples Federico II, Italy). The following experiments were carried out in laboratory: Colony interaction and competition were analysed in dual culture method (Chand & Logan 1984) in Petri plates (85 mm Ø) containing 20 ml of PDA. An antagonist plug (Byssochlamys nivea or Scopulariopsis brumptii) of 5 mm Ø was added 24 h before the Phytophthora plug in opposite position in the Petri plate. The diameter of the colonies was measured after 7 d. The volatile organic compounds (VOC) were analysed measuring the radial extension of the colonies in accordance with Dennis & Webster (1971). Briefly, a plug of B. nivea or S. brumptii (5 mm Ø) was added 24 h before the Phytophthora plug in Petri plates were overturned, overlapped and closed with parafilm. The diameter of the colonies was measured after 7 d. The weight of Phytophthora fungal mat was measured in liquid culture by incubating in flasks containing 100 ml of PDB, one plug of pathogen and one plug of antagonist at 25 C in the dark for 10 d. The Phytophthora fungal mat was separated from the PDB by filtration using a Whatman Filter MN 640 d e 110 mm Ø (Macherey-Nagel). After that, the mycelium was washed with distilled water, dried in oven at 50 C for 24 h and weighed. The controls were carried out by placing only a plug of pathogens in Petri dishes and flasks. Greenhouse tests were carried out on the experimental farm of the Plant Pathology division, Department of Agriculture, University of Naples Federico II, Italy. The biocontrol assays were conducted to assess the antagonistic effect of B. nivea and S. brumptii against P. cambivora and P. cinnamomi on Castanea sativa one-year-old seedlings. The suspensions of antagonist/pathogen strains were prepared blending the microorganisms taken from 7-day-old PDA agar plates and 500 ml of sterilized distilled water. The Colony Forming Unit (CFU) suspensions used in these experiments were adjusted to appropriate concentrations with the help of a hemocytometer and a microscope. The final suspensions of antagonists and pathogens were 1 108 CFU ml1. A number of 70 C. sativa seedlings were placed in black plastic pots (24 cm Ø) containing sterilized soils, which were achieved by autoclaving twice at 121 C for 60 min, to obtain the following treatments: a) ten chestnut plants with 10 ml of suspension of P. cambivora and 10 ml of sterilized water (control); b) ten chestnut plants with 10 ml of suspension of P. cinnamomi and 10 ml of sterilized water (control); c) ten chestnut plants with 20 ml of sterilized water (control); d) ten chestnut plants with 20 ml of suspension of B. nivea and P. cambivora; d) ten plants with 20 ml of suspension of B. nivea and P. cinnamomi; e) ten chestnut plants with 20 ml of suspension of S. brumptii and P. cambivora and; f) ten chestnut plants with 20 ml of suspension of
L. Bosso et al.
B. nivea and P. cinnamomi. The antagonists were inoculated 24 h before of the pathogens. All microorganisms used in these experiments were added around the chestnut roots. The percentage of survival plants was recorded after 1, 2, and 3 y. Koch’s postulates were applied to dead chestnut plants to detect the presence of P. cambivora or P. cinnamomi in plant tissues. Regarding the fungal antagonist, we isolated B. nivea and S. brumptii from soil according to a slightly modified protocol of Martin (1950). Briefly, 1gr of soil sample was suspended in 100 ml of distilled sterile water to make microbial suspensions (101 to 107). Dilutions between 101 and 105 were used to isolate fungi. An amount of 1 ml of microbial suspension of each concentration was added to sterile Petri dishes containing 20 ml of PDA. One percent of streptomycin solution or 1 ml of lactic acid (diluted to 20 %) was added to the medium to prevent bacterial growth. The Petri dishes were then incubated at 28 C in the dark and were observed everyday up to 5 d. The fungal strains isolated from soil were morphologically identified.
Statistical analysis All statistical analyses were carried out using XLSTAT version 2013.1. Analyses of variance (ANOVA) followed by a Tukey’s test at p-value < 0.05 were used to determine significant differences among means. All experiments were performed in triplicate.
Results Sensitivity test to PCP The fungal radial growth and hyphal thickness showed a significant decrease only at 25 mg PCP L1 for Byssochlamys nivea, approximately about 25 % and 40 %, respectively. The daily mycelium growth rate of B. nivea considerably decreased at increasing PCP concentration until it reached a reduction of 50 %. Instead, Scopulariopsis brumptii suffered a reduction of daily mycelium growth rate when the PCP concentration was of 12.5 mg L1. The spore production drastically decreased (>80 %) when PCP concentration was 25 mg L1 for both fungi (Table 1). The fungal mat also suffered a significant decrease when PCP was of 12.5 mg L1 for both fungi (>50 %). S. brumptii suffered a reduction of the fungal mat about 78 % at 25 mg L1 PCP. Both the fungi did not grow when PCP was used as sole carbon source i.e. the fungal strains produce no fungal mat (Table 1). The production of patulin significantly decreased, by less 40 % at 12.5 mg PCP L1 and by over 60 % in presence of 25 mg PCP L1 for both fungi (Table 1).
PCP removal capacity The trends of PCP adsorption and degradation showed no significant differences between the two fungi (Table 1). Byssochlamys nivea was able to adsorb 2.4 % and 4.4 % of 12.5 and 25 mg PCP L1, while Scopulariopsis brumptii adsorbed 4.8 % and 3.2 % at the same concentrations. The degradation of PCP was >95 % at any concentration of the contaminant analysed for both
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
B. nivea and S. brumptii like PCP degraders and fungal antagonists
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Table 1 e PCP sensitivity, adsorption and degradation by B. nivea and S. brumptii mycelium at different PCP concentrations. 12.5 mg PCP L1
Without PCP B. nivea Radial mycelium extension (mm) Daily mycelium growth rate (mm day1) Hyphal size (mm) Spore production (x 1 250 000) Fungal mat (mg) Fungal mat with PCP as sole carbon source (mg) Patulin production (mg L1) PCP adsorption (mg) PCP degradation (mg)
aA
85.0 0.0 10.6 0.0aA 5.5 0.2aA 666.5 7.8aA 184.4 7.0aA 94.0 1.2aA 15.9 0.1aA e e
S. brumptii aA
85.0 0.0 10.5 0.1aA 7.2 1.2bA 225.5 0.7bA 218.7 2.0bA 95.3 0.2bA 12.4 0.2bA e e
B. nivea aA
83.1 2.4 7.5 0.2aB 5.3 0.3aA 640.5 13.0aA 51.95 9.0aB 0.0 0.0aB 10.2 0.2aB 0.30 0.2aA 12.1 0.3aA
25 mg PCP L1
S. brumptii aA
85.0 0.0 7.7 0.1aB 7.2 0.9bA 126.5 2.1bB 106.33 8.0bB 0.0 0.0aB 8.3 0.1bB 0.60 0.2aA 11.8 0.2aA
B. nivea
S. brumptii aB
64.8 17.5 5.8 1.5aC 3.2 0.9aB 58.1 4.2aB 50.27 5.0aB 0.0 0.0aB 6.1 0.1aC 1.10 0.4aB 23.8 0.4aB
83.3 2.5bA 7.5 0.2bB 7.2 0.7bA 43.3 1.4bC 27.7 7.0bC 0.0 0.0aB 4.3 0.1aC 0.80 0.2aA 24.1 0.2aB
Different lower case letters (a and b) refer to significant differences (P < 0.05) between PCP sensitivity, adsorption and degradation of B. nivea and S. brumptii at the same PCP concentration. Different upper case letters (A, B and C) refer to significant differences (P < 0.05) among PCP sensitivity, adsorption and degradation of B. nivea and S. brumptii at different PCP concentration.
fungi in liquid culture (Table 1). In the control, without fungal mycelium, the PCP removed was negligible.
Electrolyte Leakage Assay
and 3 y, respectively. Finally, only 60 % of plants used for control were found alive after 1 y, while, all chestnut plants died after 3 y in treatments with only the pathogens without the antagonist (Table 2). P. cambivora and P. cinnamomi were the
The conductance of all samples increased over time reaching a stable value after 7 h (Fig 1). The incubation of Byssochlamys nivea and Scopulariopsis brumptii with PCP did not produce any PCP toxic effects on mycelium without significant variation of electrolyte leakage after 24 h (Fig 1a,b). The experiment carried out on vegetable tissues highlighted that the conductance of all systems increased by about 80 % over time (Fig 2) and no significant effect of fungal metabolites on tomato stems was ascertained at 24 h. However, an increase of electrolyte leakage of the tomato stem was registered during the first eight hours in the presence of B. nivea and in the range of 2e5 h in the presence of S. brumtii respect to control.
Antagonistic assays of Byssochlamys nivea and Scopulariopsis brumptii versus Phytophthora cambivora and Phytophthora cinnamomi under laboratory and greenhouse conditions Radial mycelium extension was measured on PDA in colony interactions and VOC experiments. In the first case, the growth of Phytophthora cambivora and Phytophthora cinnamomi was reduced to 40 % and 60 % when these pathogens were cultured with Scopulariopsis brumptii and Byssochlamys nivea, respectively (Table 2). The radial growth of Phytophthora isolates, measured in VOC experiments in presence of the antagonist fungi, showed no significant difference respect to the control (Table 2). Finally, S. brumptii and B. nivea reduced to 50 % and 60 % the fungal mat production of P. cambivora and P. cinnamomi in flask (Table 2). In greenhouse experiments, the survival of chestnut plants was recorded annually for three years. The highest percentage of plants survived was found in the treatment with S. brumptii (about 70 %) against both P. cambivora and P. cinnamomi until the end of the experiment (Table 2). The plants survived in the treatment with B. nivea were about 70 % and 50 % after 1
Fig 1 e Conductance values (mean ± standard deviation) during 24 h of incubation using Electrolyte Leakage Assay (ELA) of: a) B. nivea incubated in distilled water (white bar) and B. nivea incubated at 25 mg PCP LL1 (black bar); b) S. brumptii incubated in distilled water (light grey bar) and S. brumptii incubated at 25 mg PCP LL1 (dark grey bar). Different small letters (a and b) refer to significant differences (P < 0.05) among PCP sensitivity. The conductance values were calculated as the difference from the reading at the beginning of the assay (increments in microsiemens from t0).
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
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L. Bosso et al.
Fig 2 e Conductance values (mean ± standard deviation) of tomato stems in the presence of B. nivea (black bars) and S. brumptii (grey bars) culture filtrates during 24 h of incubation using Electrolyte Leakage Assay (ELA). Different small letters (a, b and c) refer to significant differences (P < 0.05) among treatments at the same time. The conductance values were calculated as the difference from the reading at the beginning of the assay (increments in microsiemens from t0).
cause of dead chestnut plants in accordance with Koch’s postulates.
Discussion Byssochlamys nivea and Scopulariopsis brumptii in PCP sensitivity The fungal strains showed a good PCP sensitivity at the concentrations tested. In Petri dishes, Byssochlamys nivea suffered a decrease of radial mycelium extension, hyphal thickness and daily mycelium growth rate in the presence of 25 mg PCP L1. Instead, Scopulariopsis brumptii grew without significant limitations due to PCP. A strain of Rhizopus nigricans was able to grow even at 100 mg PCP L1 while no growth was detected at 250 mg PCP L1 (Tomasini et al. 2001). Moreover the R. nigricans mycelium daily growth rate was 9.8 and 9.6 mm day1 with 12.5 and 25 mg PCP L1, respectively. Our fungi were slower in growth (in average 7 mm day1 for both
fungi) than R. nigricans, but, after 5 d, the radial mycelium extension of our fungi was 55 mm against to 45 mm of R. nigricans. The radial growth of Aspergillus niger was completely inhibited at 40 mg PCP L1, while at concentrations of 10 and 20 mg PCP L1, the mycelium growth was reduced to 75 % and 88 %, respectively (Bomar & Bomar 1999). At the same PCP concentrations, A. niger formed only vegetative structures without conidia formation. B. nivea and S. brumptii did not suffer of the same radial extension reduction, although, the spore production decreased by increasing PCP concentration. After the experiment the conidia were still able to germinate for both fungi. Phanerochaete chrysosporium showed a daily mycelium growth rate of 10.3 cm day1 at 12.5 mg PCP L1, but its growth rate decreased until 8.2 cm day1 by increasing PCP concentration at 25 mg PCP L1 (Tomasini et al. 1996). Respect to P. chrysosporium, our fungal strains showed the same daily mycelium growth rate at 12.5 mg PCP L1, while they were slower than P. chrysosporium at 25 mg PCP L1. White-rot Chilean isolates were tested in resistance and mycelium radial extension at 25 mg PCP L1 in N-limited solid media (Tortella
Table 2 e Antagonistic assays of B. nivea and S. brumptii against P. cambivora and P. cinnamomi under laboratory and greenhouse conditions. Without fungal antagonist P. cambivora Colony interaction (mm) Volatile organic compounds (mm) Fungal mat (mg) Chestnut live plants after 1 ya (%) Chestnut live plants after 2 ya (%) Chestnut live plants after 3 ya (%)
aA
42.5 0.5 42.5 0.5aA 8.7 0.5aA 60 10 0
P. cinnamomi bA
40.1 0.2 40.1 0.2bA 8.2 0.5aA 60 10 0
B. nivea P. cambivora aB
12.3 0.2 41.3 0.8aA 2.9 0.5aB 72 60 50
S. brumptii
P. cinnamomi aB
12.8 0.5 40.1 0.6aA 3.0 0.2aB 65 65 50
P. cambivora aC
24.2 0.6 41.3 0.8aA 5.2 0.5aC 75 70 70
P. cinnamomi 23.2 0.8aC 40.3 0.6aA 5.6 0.3aC 75 75 70
Different lower case letters (a and b) refer to significant differences (P < 0.05) between growth values of P cambivora and P. cinnamomi versus the same fungal antagonist. Different upper case letters (A, B and C) refer to significant differences (P < 0.05) among growth values of P cambivora and P. cinnamomi versus different fungal antagonist. a Greenhouse experiments.
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
B. nivea and S. brumptii like PCP degraders and fungal antagonists
et al. 2008). When the fungi were exposed to PCP, only Lenzites betulina had a growth rate of 10 mm day1 similarly to our fungi tested in this study. Patulin and fungal mat production were negatively influenced by increasing PCP concentration. In literature there are no studies on the interaction between the patulin production and PCP. B. nivea and S. brumptii reduced by 60 % the patulin production at 25 mg PCP L1. Same results were obtained when the fungi were grown in PDB to evaluate the fungal mat. Fungal strains were cultured for 7 d with 25 mg PCP1 in liquid batch and they produced a reduction in growth of the fungal mat by 74 % in Volvariella volvacea; 77 % in Armillaria gallica; 30 % in Armillaria mellea; 28 % in Ganoderma lucidum and 17 % in Pleurotus pulmonarius (Chiu et al. 1998). In cultures with 25 and 12.5 mg PCP L1, the fungal mat growth of Amylomyces rouxii was 8 mg after 96 h (Marcial et al. 2006). In latter case the fungal mat growth was significantly lower than our fungi. When B. nivea and S. brumptii were cultured in liquid mineral medium with PCP as sole carbon source, the fungi showed no fungal mat production. Instead when glucose was added in the flask, fungal mat was achieved for both fungi. This essentially happened because, unlike bacteria, fungi do not utilize PCP as a source of carbon and energy (McAllister et al. 1996). In fungi the degradation of PCP is not consequence of enzyme tools which are able to do this function but an effect of cometabolism (McAllister et al. 1996; Bosso & Cristinzio 2014). The effects of PCP on hyphal size, mycelium morphology and spore production of B. nivea and S. brumptii were made more evident by increasing PCP concentration, although the PCP never reduced u the fungal growth of B. nivea and S. brumptii to zero. ELA assays showed that B. nivea and S. brumptii, after 24 h of incubation at 25 mg PCP L1, did not suffer a decrease in electrolyte leakage compared to control incubated in distilled water. It is widely known that PCP can negatively influence fungi cellular processes, morphology, lipid membrane components, fungal mat growth, enzymatic activity, sporulation and reproduction capacity (Watanabe 1978; Bajpaia & Banerjib 1992) but, in our case, B. nivea and S. brumptii seem to have a good sensitivity to PCP. Sensitivity to PCP represents the most important characteristic required to select specific and effective microorganisms useful in PCP degradation.
Efficiency of Byssochlamys nivea and Scopulariopsis brumptii in PCP removal Fresh mycelium of Byssochlamys nivea and Scopulariopsis brumptii was able to adsorb and degrade PCP. While for both fungi the adsorption was <5 %, B. nivea and S. brumptii degraded about 95 % of the PCP after 28 d of incubation (Table 1). These fungal strains are known to not produce extracellular enzymes as lignin peroxidases or laccase which are highly efficient in degrading PCP in co-metabolism (McAllister et al. 1996; Bosso & Cristinzio 2014). However, S. brumptii produce phenoloxidase enzymes (Tanaka et al. 2000) which can be useful in PCP degradation (Bosso et al. 2015b). B. nivea was able to degrade 12.5 and 25 mg PCP L1 after 28 d in flask due to the pectinolytic enzymes, which are widely used in bioremediation strategy (Chiu & Chang 1973; Bosso et al. 2011; Bosso et al. 2015b). In fact, in Byssochlamys genus
7
some enzymes were detected useful in the degradation pathway of lignin and other wood constituents (Chiu & Chang 1973: Furukawa et al. 1999). The ability of B. nivea and S. brumptii to remove PCP was excellent after 28 d of incubation. A strain of Byssochlamys fulva was able to remove 20 % of 50 mg PCP L1 in only 8 d (Scelza et al. 2008). Excellent results in PCP depletion were also obtained with an isolate of Rhizopus nigricans (Tomasini et al. 2001). In fact the strain degraded 12.5 mg PCP L1 using phenoloxidase activity in only 8 d. In N-limited liquid medium Anthracophyllum discolor, Lenzites betulina and Galerina patagonica removed 25 mg PCP L1 in only 15 d thanks to manganese and lignin peroxidase production (Tortella et al. 2008). The maximum efficiency in PCP removal was obtained by Ganoderma lucidum, Phanerochaete chrysosporium and Polyporus sp. after 7 d of incubation in liquid batch system with 25 mg PCP L1. These fungi were able to remove 75e78 % of the PCP initial concentration (Chiu et al. 1998). The majority of fungi implicated in PCP degradation are members of the white-rot Basidiomycetes and are capable of degrading lignin. Almost all studies on PCP degradation were conducted with P. chrysosporium, Phanerochaete sordida, Trametes versicolor and Trametes hirsuta (McAllister et al. 1996; Bosso & Cristinzio 2014). Few studies about PCP removal by Ascomycota fungi are available in literature (Rubilar et al. 2012; Bosso et al. 2015a). This justifies the importance to study fungi belonging to this fungal group that may tolerate and degrade high PCP concentrations.
Biocontrol of Phytophthora cambivora and Phytophthora cinnamomi by Byssochlamys nivea and Scopulariopsis brumptii Byssochlamys nivea and Scopulariopsis brumptii have demonstrated excellent capacity to reduce the growth of Phytophthora cambivora and Phytophthora cinnamomi in all microbiological media. Several antagonists and their metabolites were found useful in Phytophthora’s control. A Flufluran derivate extract by Aspergillus flavus completely inhibited the mycelium growth of P. cinnamomi after 7 d (Evidente et al. 2009). Antibiosis and mycoparasitism action by some fungi and bacteria, isolated by manure compost, were able to lyse Phytophthora’s mycelium with inhibition values between 40 and 70 %. b-glucosidase and phosphates activities of a Trichoderma sp. strain reduced the development of P. cinnamomi (Kelley & Rodriquez-Kabana 1976). A methanolic compound obtained from the cyanobacterium Nostoc was tested with good results against a variety of pathogens of agricultural importance among which: P. cambivora and P. cinnamomi (Aryantha & Guest 2006). Antibiosis between our fungi and Phytophthorae was due essentially to the patulin. Patulin is a mycotoxin with antagonist capacity against a wide spectrum of microorganisms. Patulin produced by Aspergillus clavatonaticus exhibited inhibitory activity in vitro against several plant pathogenic fungi i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum, Rhizoctonia solani and Pythium ultimum (Zhang et al. 2008). This mycotoxin is able to stop the synthesis of rRNA, tRNA and mRNA in Saccharomyces cerevisiae (Sumbu et al. 1983) and to have antibacterial activities against Escherichia coli and Micrococcus luteus (Praveena & Padmini 2011). P. cambivora and P. cinnamomi showed no significant decrease in radial growth when cultured in VOC experiments.
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
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The VOCs are often used in Phytophthora control but our experiments not appear effective to limit the growth of the pathogens. The endophytic fungus Muscodor crispans produced VOCs that have inhibitory effects against P. cinnamomi, P cambivora and other oomycetes such as Phytophthora palmivora, P. ultimum (Mitchell et al. 2010). Furthermore Nodulisporium sp., another endophytic fungus, produces VOCs inhibitors against a number of pathogens such as Aspergillus fumigatus and R. solani, P. cinnamomi and Sclerotinia sclerotiorum within 48 h of exposure (Mends et al. 2012). The potential of B. nivea and S. brumptii as biocontrol agents to suppress P. cinnamomi and P. cambivora was evaluated in greenhouse experiments. B. nivea and S. brumptii had a biocontrol on Phytophthora species. No chestnut plants survived in the control after 3 y. On the other hand, we obtained a survival of chestnut plants >50 % in the treatments with B. nivea and S. brumptii (Table 2). Several microorganism antagonists to P. cambivora and P. cinnamomi have been described in literature, providing considerable scientific interest, as well as: Trichoderma harzianum, Bacillus subtilis, Penicillium funiculosum and Gliocladium virens (Howell 2003; Sylvia et al. 2005; Griffin 2014). Although antibiosis seems to be the strategy involved in the biocontrol activity by B. nivea and S. brumptii, further analysis of their modes of action are currently being undertaken in order to improve their use and implementation under real culture conditions. Biocontrol by B. nivea and S. brumptii of P. cambivora and P. cinnamomi seems to be a good alternative for sustainable agriculture to overcome the problems of public concern associated with pesticides and pathogens resistant to chemical pesticides. Ink disease, caused by the oomycete pathogens P. cambivora and P. cinnamomi, is probably the most prevalent Chestnut disease. It is very destructive and causes flame shaped dark necroses on collar rot of adult trees, shrubs and seedlings. Biological control is an excellent alternative for sustainable agriculture to avoid problems associated with the use of pesticides. Many studies have been done to date; although almost always conducted at laboratory scale. In this regard, B. nivea and S. brumptii do not produce toxic compounds able to damage cell walls and membranes of tomato stem cells; as shown in the ELA experiment (Fig 2). In contrast, the incubation of tomato stems in A. flavus culture filtrates produced high phytotoxicity due probably to hydrophilic metabolites that remainin the aqueous phase (Evidente et al. 2009). In our experiments no significant difference in conductance was detected between the tomato stem incubated in distilled water and in B. nivea or S. brumptii culture filtrates. The next research step is to address in a field study the results obtained in this research.
Conclusions Critical analysis reveals that there are relatively few reports on the use of microorganisms in the double role of bioremediation and biocontrol agent. This is the first paper about the capacity of Byssochlamys nivea and Scopulariopsis brumptii to remove PCP and to control the Phytophthora species agents of Ink disease on chestnut. Furthermore, the excellent result showed by ELA assays on tomato stems indicate that our fungi do not cause significant damage to plants and that they can be
L. Bosso et al.
used in field experiments. For this reason, our results indicate that B. nivea and S. brumptii have an interesting potential to be used in bioremediation and biocontrol strategy.
Acknowledgements We thank Prof Dianna Pickens for the linguistic revision.
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Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species Luciano BOSSOa,*, Rosalia SCELZAa, Rosaria VARLESEa, Giuseppe MECAb, Antonino TESTAa, Maria A. RAOa, Gennaro CRISTINZIOa n. 100, 80055 Portici (Naples), Italy Department of Agriculture, University of Naples Federico II, via Universita Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjassot, Spain
a
b
article info
abstract
Article history:
Bioremediation and biological-control by fungi have made tremendous strides in numer-
Received 8 May 2015
ous biotechnology applications. The aim of this study was to test Byssochlamys nivea and
Received in revised form
Scopulariopsis brumptii in sensitivity and degradation to pentachlorophenol (PCP) and in bi-
5 January 2016
ological-control of Phytophthora cinnamomi and Phytophthora cambivora. B. nivea and S.
Accepted 6 January 2016
brumptii were tested in PCP sensitivity and degradation in microbiological media while
Corresponding Editor:
the experiments of biological-control were carried out in microbiological media and soil.
Nabla Kennedy
The fungal strains showed low PCP sensitivity at 12.5 and 25 mg PCP L1 although the hyphal size, fungal mat, patulin, and spore production decreased with increasing PCP concen-
Keywords:
trations. B. nivea and S. brumptii depleted completely 12.5 and 25 mg PCP L1 in liquid
Bioremediation
culture after 28 d of incubation at 28 C. Electrolyte leakage assays showed that both fungi
Electrolyte Leakage Assay
have low sensitivity to 25 mg PCP L1 and produced no toxic compounds for the plant. B.
Fungi
nivea and S. brumptii were able to inhibit the growth of the two plant pathogens in labora-
Oomycetes
tory studies and reduce the mortality of chestnut plants caused by two Phytophthorae in
Patulin
greenhouse experiments. The two fungal strains did not produce volatile organic com-
Plant pathogens
pounds able to reduce the growth of two plant pathogens tested. ª 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Bioremediation is defined as the process whereby living organisms are able to transform environmental contaminants into less toxic forms. It uses naturally occurring bacteria, fungi or plants to degrade or detoxify substances hazardous
to human health and/or the environment (Crawford & Crawford 1997; Vidali 2001; Olguin 2003; Petroselli et al. 2014). Biological control (biocontrol) refers to the purposeful utilization of introduced or resident living organisms to suppress the activities and populations of one or more plant
* Corresponding author. Tel.: þ39 081 2539371, þ39 3290758021(mobile). E-mail address: [email protected] (L. Bosso). http://dx.doi.org/10.1016/j.funbio.2016.01.004 1878-6146/ª 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
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pathogens to improve plant health. Disease suppression by biocontrol agents is the sustained manifestation of interactions among the plant, the pathogen, the antagonist, the microbial community on and around the plant, and the physical environment (Handelsman & Stab 1996; Howell 2003; Lorito et al. 2010). The use of beneficial fungi and bacteria for bioremediation and biocontrol of plant pathogens has made tremendous strides in numerous biotechnology applications. In recent years the need to find a global approach to environmental and agricultural issues has set the challenge to discover microorganisms which are useful for both bioremediation and biocontrol research (Griffin 2014). This approach is primarily focused on how to use microorganism strains to inhibit the dangerous advance of plant pathogens and to deplete environmental contaminants (Sylvia et al. 2005; Singh et al. 2011). The fungi that play important roles in biocontrol and bioremediation strategies are numerous; some of these are grouped as mycorrhizal and endophytic fungi (Griffin 2014). The endophytic fungus Phomopsis sp. has been found to use the 4-hydroxybenzoic acid as the only carbon source (Chen et al. 2011) and to inhibit the growth of Physocnemum brevilineum which is a vector of Ceratocystis ulmi, an important elm pathogen (Webber 1981). The fungi belonging to genus Glomus are arbuscular mycorrhizas used in the biocontrol n-Aguilar & Barea 1996) of soil-borne plant pathogens (Azco and as heavy metal biosorbents in soil (Leyval et al. 1997). Filamentous fungal species belonging to the genus Trichoderma are able to counteract some plant pathogens by means of mycoparasitism and antibiosis (Howell 2003: Lorito et al. 2010) and simultaneously to deplete pollutants including heavy metals, pentachlorophenols (Bosso & Cristinzio 2014) and polycyclic aromatic hydrocarbons (PAHs) (Tripathi et al. 2013). Aspergillus flavus was used as a biosorbent of heavy metals (Deepa et al. 2006), phenol degrader (Ghanem et al. 2009; Bosso & Cristinzio 2014) and promoter of Phytophthorae’s growth inhibition (Evidente et al. 2009). Penicillium spp. has demonstrated an excellent ability to degrade different xenobiotic compounds such as phenolic compounds, ~ o 2009). Nevertheless some PAHs and heavy metals (Leita strains of genus Penicillium were also used in biocontrol. In fact, Penicillium funiculosum and Penicillium janthinellum have been able to limit the Phytophthorae root rots of azalea (Ownley & Benson 1992; Fang & Tsao 1995). Some species of genus Verticillium have been found to remove petroleum products and PAHs in soil (Gadd 2001; Singh et al. 2011) and to control numerous plant pathogens such as fungi, bacteria rillon & Ramawat 2012). Other interestand nematodes (Me ing ecological groups are for example the white-rot fungi. Trametes versicolor has shown promising biocontrol activities ~ as & Martınez 1996) against Fusarium oxysporum (Ruiz-Duen and represents one of the most important organisms used in bioremediation actions (Gadd 2001; Singh 2006; Bosso & Cristinzio 2014). Pentachlorophenol (PCP) is a toxic compound which is widely used as a wood treatment agent and general biocide. It is persistent in the environment and has been classified as a priority contaminant to be reclaimed in many countries. In fact, uncontrolled PCP uses and releases have
L. Bosso et al.
caused contamination of soil, water and ground water. Although PCP is recalcitrant to biodegradation, numerous bacterial and fungal isolates have been reported to be able to degrade it (Gadd 2001; Singh 2006; Bosso & Cristinzio 2014). In the present study, two fungal strains which were isolated from artificially PCP-contaminated soil during a longterm bioremediation study and identified as Byssochlamys nivea (Westling 1909) and Scopulariopsis brumptii (SalvanetDuval 1935) (Scelza et al. 2008; Bosso et al. 2011; Bosso et al. 2015b), were tested in laboratory and greenhouse experiments to reach the following goals: 1 to evaluate their sensitivity to PCP and their capacity for contaminant degradation; 2 to assess their antagonistic effect vs Phytophthora cinnamomi (Rands 1922) and Phytophthora cambivora (Buisman 1927), soil-borne pathogens causing disease on many woody hosts (root and collar rot of adult trees and of seedlings in nurseries, plantations and forests) especially on plants belonging the genus Castanea (Vannini & Vettraino 2001).
Materials and methods Fungal strains and cultivation conditions Byssochlamys nivea and Scopulariopsis brumptii were stored into slant tubes containing potato dextrose agar (PDA; 5 g L1 potato; 20 g L1 dextrose; 15 g L1 agar) at 20 C at the laboratories of Forest Pathology of the Department of Agriculture (University of Naples Federico II, Italy).
Chemicals PCP (>99 % purity) was purchased from SigmaeAldrich (USA). All solvents and chemicals reagents were purchased from Carlo Erba Reagents (Italy).
Sensitivity test to PCP in plate culture Fungal sensitivity to PCP was evaluated in Petri dishes. The fungal strains were grown onto PDA at different PCP concentrations (12.5 and 25 mg L1) prepared previously by dissolving the suitable amounts of PCP in 5 ml of methanol and then in PDA (final volume 1 L). This step was necessary because the PCP is weakly insoluble in water. The controls were cultured on PDA having 5 ml L1 of methanol but without PCP. All samples were incubated at 25 C for 7 d. Sensitivity to PCP was determined by measuring the diameter (Ø) of the colony, daily mycelium growth rate (Tomasini et al. 2001), hyphal size (this was achieved measuring with an ocular micrometer at total magnification of 100 the hyphal thickness in mm) and spore production (St-Arnaud et al. 1996). A number of 100 measurements were carried out for each replicate to determine hyphal thickness and spore production.
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B. nivea and S. brumptii like PCP degraders and fungal antagonists
Sensitivity test to PCP and PCP removal in liquid culture Fungal sensitivity to PCP was determined also in liquid culture. Two plugs of 8 mm Ø of active mycelia were cut from PDA cultures of 7 d old and were grown in potato dextrose broth (PDB; 5 g L1 potato; 20 g L1 dextrose) with 12.5 or 25 mg PCP L1, prepared as described above. The controls were cultured without PCP. Cultures were grown in 250 ml Erlenmeyer flask containing 100 ml of medium. All samples were incubated under shaking conditions at 120 rpm for 28 d at 28 C. Sensitivity test was evaluated measuring the dry weight fungal mat. The fungal mat was separated from PDB by filtration using Whatman Filter MN 640 d e 110 mm Ø (Macherey-Nagel). The fungal mat was washed with distilled water, dried in an oven at 50 C for 24 h and weighed. PCP concentration in the fungal mat and liquid medium was determined by HPLC in according to the methods described in Tomasini et al. (2001). Briefly, the samples were filtered and the biomass was put on a sonic bath for 10 min using a carbonate buffer, pH 11. These fractions and similarly the culture broth samples were filtered through a 0.22 mm Whatman membrane (Macherey-Nagel). PCP from each fraction, biomass and culture broth, was quantified by HPLC and these two values were as added to the total residual PCP. Samples were analysed by HPLC with a C-18 reverse column (mbondapack Waters) packed with R-Sil C-18 (18 mm). Mobile phase was 1 % acetic acid water solution and 1 % acetic acid acetonitrile solution (75:25) at a flow rate of 0.8 ml min1. Elution was monitored at 238 nm and quantified with an integrator (Waters).
Fungal growth in presence of PCP as sole carbon source The fungal strains were grown in liquid mineral medium enriched with PCP as the sole carbon source. Each fungus was inoculated into a flask containing 12.5 or 25 mg PCP L1 in a medium having the following composition (g L1): KH2PO4 (2.0); MgSO4$7H2O (0.5); CaCl2 (0.1); NH4Cl (0.12); ZnSO4$7H2O (0.1); MnCl2$4H2O (0.3). The control was obtained with the same mineral medium but having 10 g L1 of glucose as the carbon source. The medium pH was adjusted to 5.5 before sterilization. The flasks were inoculated with two plugs of 8 mm Ø of active mycelia taken from 7-day-old PDA cultures. The incubation was carried out on orbital shaker incubator at 120 rpm in the dark for 28 d at 28 C. After that, the dry weight of the fungal mat was measured as described above. The analytical methods used to determine the PCP concentration in the fungal mat and in the liquid mineral medium were extensively described in the previous paragraph.
Production of patulin in liquid culture enriched by PCP For patulin extraction, the method of Raiola et al. (2012) was applied. In particular, 10 ml of liquid culture of Byssochlamys nivea or Scopulariopsis brumptii, obtained after 28 d of incubation in PDB, were transferred into a 50 ml-plastic tube containing 15 g of Na2SO4, 2 g of NaHCO3 and 10 ml of extraction solution (ethyl acetate/hexane 60:40, v/v) were added. The tube was shaken for 3.5 min on a mechanical
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shaker (Inter-Continental equipment, Hidalgo, TX) and then centrifuged at 2000 rpm for 1 min at 4 C. The supernatant (2.5 ml) was immediately placed onto an unconditioned Strata C18-E solid phase extraction column (Phenomenex, USA) which was then washed with 3 ml of the extraction solution adjusting the flow to one drop per second by using slight air pressure. The eluate was collected in a 10 ml-plastic test tube containing 50 mL of acetic acid. The solvent was evaporated at max 40 C for about 35 min in a vacuum centrifuge Thermo Savant (Speed Vacuum Thermo Electron Corporation Milford, MA, USA) and 1 ml of acidulated water (pH 4) was used to dissolve the sample. The solution was mixed with a vortex (Biosan MSV-3500, Lietsa, Finland) filtered with 0.22 mm filters (Phenomenex, Palo Alto, CA) and injected in the HPLC apparatus (Shimadzu-Japan) equipped with an autosampler SIL-20A, two pumps LC-20AD and a UV/vis detector SPD-20A set at 276 nm wavelength. The column was a Gemini 5 mm C18, 110 A (150 mm 2 mm) (Phenomenex, CA, USA). The mobile phase was water containing 1 % acetic acid (A) and methanol (B) (95/5, v/v) in isocratic conditions. The flow rate was of 1 ml min1. Under these chromatographic conditions, the retention time for patulin was 14.0 0.1 min.
Electrolyte Leakage Assay (ELA) Electrolyte Leakage Assay (ELA) is a test that assesses the toxicity in plant tissues, as this toxicity is linked to the damage on plant cell wall and membrane induced by toxic compounds (Peever & Higgins 1989). ELA was carried out on both fungi to assess: i) the toxic effect of PCP on mycelium and; ii) the potential damage on plant cell wall and membrane due to fungal metabolites. Regarding the first purpose, six plugs of 5 mm Ø of active mycelia were taken from 7-day-old water agar cultures (WA, Carlo Erba) and incubated in the dark for 24 h in beakers containing a water solution of 25 mg PCP L1. The control was prepared incubating the fungus with only distilled water. After the incubation all fungal plugs were washed and transferred into a beaker containing 15 ml of distilled water. The ELA measurements were conducted after 0.5, 1, 2, 4, 7, 8, and 24 h. For the second purpose, ten tomato (Solanum lycopersicum) stem pieces (5-mm long) were incubated overnight in a culture filtrate of Byssochlamys nivea or Scopulariopsis brumptii. The fungal culture filtrate was obtained from PDB medium with active mycelia cultured for 7 d. The culture filtrate was separated from the mycelium by filtration with Whatman Filter MN 640 d e 110 mm Ø (Macherey-Nagel). The control was obtained by incubating the stem in only PDB. After the incubation, all the stem pieces were washed and transferred into a beaker containing 15 ml of distilled water. The measurements were set hourly except for the first and last analyses which were made after 0.5 and 24 h, respectively. In all the experiments, the conductance was measured in microsiemens (mS) using a conductivity-meter with 20 electrodes (range 20e200 mS cm1; K ¼ 1). The conductance values were calculated as the difference from the reading at the beginning of the assay (increments in microsiemens from t0) (Evidente et al. 2009).
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
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Antagonistic assays of Byssochlamys nivea and Scopulariopsis brumptii versus Phytophthora cambivora and Phytophthora cinnamomi under laboratory and greenhouse conditions The antagonistic assays were carried out among our fungal strains against Phytophthora cambivora and Phytophthora cinnamomi under laboratory and greenhouse conditions. These two Chromisti are stored into PDA slant tubes at 18 C at the laboratories of Forest Pathology of the Department of Agriculture (University of Naples Federico II, Italy). The following experiments were carried out in laboratory: Colony interaction and competition were analysed in dual culture method (Chand & Logan 1984) in Petri plates (85 mm Ø) containing 20 ml of PDA. An antagonist plug (Byssochlamys nivea or Scopulariopsis brumptii) of 5 mm Ø was added 24 h before the Phytophthora plug in opposite position in the Petri plate. The diameter of the colonies was measured after 7 d. The volatile organic compounds (VOC) were analysed measuring the radial extension of the colonies in accordance with Dennis & Webster (1971). Briefly, a plug of B. nivea or S. brumptii (5 mm Ø) was added 24 h before the Phytophthora plug in Petri plates were overturned, overlapped and closed with parafilm. The diameter of the colonies was measured after 7 d. The weight of Phytophthora fungal mat was measured in liquid culture by incubating in flasks containing 100 ml of PDB, one plug of pathogen and one plug of antagonist at 25 C in the dark for 10 d. The Phytophthora fungal mat was separated from the PDB by filtration using a Whatman Filter MN 640 d e 110 mm Ø (Macherey-Nagel). After that, the mycelium was washed with distilled water, dried in oven at 50 C for 24 h and weighed. The controls were carried out by placing only a plug of pathogens in Petri dishes and flasks. Greenhouse tests were carried out on the experimental farm of the Plant Pathology division, Department of Agriculture, University of Naples Federico II, Italy. The biocontrol assays were conducted to assess the antagonistic effect of B. nivea and S. brumptii against P. cambivora and P. cinnamomi on Castanea sativa one-year-old seedlings. The suspensions of antagonist/pathogen strains were prepared blending the microorganisms taken from 7-day-old PDA agar plates and 500 ml of sterilized distilled water. The Colony Forming Unit (CFU) suspensions used in these experiments were adjusted to appropriate concentrations with the help of a hemocytometer and a microscope. The final suspensions of antagonists and pathogens were 1 108 CFU ml1. A number of 70 C. sativa seedlings were placed in black plastic pots (24 cm Ø) containing sterilized soils, which were achieved by autoclaving twice at 121 C for 60 min, to obtain the following treatments: a) ten chestnut plants with 10 ml of suspension of P. cambivora and 10 ml of sterilized water (control); b) ten chestnut plants with 10 ml of suspension of P. cinnamomi and 10 ml of sterilized water (control); c) ten chestnut plants with 20 ml of sterilized water (control); d) ten chestnut plants with 20 ml of suspension of B. nivea and P. cambivora; d) ten plants with 20 ml of suspension of B. nivea and P. cinnamomi; e) ten chestnut plants with 20 ml of suspension of S. brumptii and P. cambivora and; f) ten chestnut plants with 20 ml of suspension of
L. Bosso et al.
B. nivea and P. cinnamomi. The antagonists were inoculated 24 h before of the pathogens. All microorganisms used in these experiments were added around the chestnut roots. The percentage of survival plants was recorded after 1, 2, and 3 y. Koch’s postulates were applied to dead chestnut plants to detect the presence of P. cambivora or P. cinnamomi in plant tissues. Regarding the fungal antagonist, we isolated B. nivea and S. brumptii from soil according to a slightly modified protocol of Martin (1950). Briefly, 1gr of soil sample was suspended in 100 ml of distilled sterile water to make microbial suspensions (101 to 107). Dilutions between 101 and 105 were used to isolate fungi. An amount of 1 ml of microbial suspension of each concentration was added to sterile Petri dishes containing 20 ml of PDA. One percent of streptomycin solution or 1 ml of lactic acid (diluted to 20 %) was added to the medium to prevent bacterial growth. The Petri dishes were then incubated at 28 C in the dark and were observed everyday up to 5 d. The fungal strains isolated from soil were morphologically identified.
Statistical analysis All statistical analyses were carried out using XLSTAT version 2013.1. Analyses of variance (ANOVA) followed by a Tukey’s test at p-value < 0.05 were used to determine significant differences among means. All experiments were performed in triplicate.
Results Sensitivity test to PCP The fungal radial growth and hyphal thickness showed a significant decrease only at 25 mg PCP L1 for Byssochlamys nivea, approximately about 25 % and 40 %, respectively. The daily mycelium growth rate of B. nivea considerably decreased at increasing PCP concentration until it reached a reduction of 50 %. Instead, Scopulariopsis brumptii suffered a reduction of daily mycelium growth rate when the PCP concentration was of 12.5 mg L1. The spore production drastically decreased (>80 %) when PCP concentration was 25 mg L1 for both fungi (Table 1). The fungal mat also suffered a significant decrease when PCP was of 12.5 mg L1 for both fungi (>50 %). S. brumptii suffered a reduction of the fungal mat about 78 % at 25 mg L1 PCP. Both the fungi did not grow when PCP was used as sole carbon source i.e. the fungal strains produce no fungal mat (Table 1). The production of patulin significantly decreased, by less 40 % at 12.5 mg PCP L1 and by over 60 % in presence of 25 mg PCP L1 for both fungi (Table 1).
PCP removal capacity The trends of PCP adsorption and degradation showed no significant differences between the two fungi (Table 1). Byssochlamys nivea was able to adsorb 2.4 % and 4.4 % of 12.5 and 25 mg PCP L1, while Scopulariopsis brumptii adsorbed 4.8 % and 3.2 % at the same concentrations. The degradation of PCP was >95 % at any concentration of the contaminant analysed for both
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
B. nivea and S. brumptii like PCP degraders and fungal antagonists
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Table 1 e PCP sensitivity, adsorption and degradation by B. nivea and S. brumptii mycelium at different PCP concentrations. 12.5 mg PCP L1
Without PCP B. nivea Radial mycelium extension (mm) Daily mycelium growth rate (mm day1) Hyphal size (mm) Spore production (x 1 250 000) Fungal mat (mg) Fungal mat with PCP as sole carbon source (mg) Patulin production (mg L1) PCP adsorption (mg) PCP degradation (mg)
aA
85.0 0.0 10.6 0.0aA 5.5 0.2aA 666.5 7.8aA 184.4 7.0aA 94.0 1.2aA 15.9 0.1aA e e
S. brumptii aA
85.0 0.0 10.5 0.1aA 7.2 1.2bA 225.5 0.7bA 218.7 2.0bA 95.3 0.2bA 12.4 0.2bA e e
B. nivea aA
83.1 2.4 7.5 0.2aB 5.3 0.3aA 640.5 13.0aA 51.95 9.0aB 0.0 0.0aB 10.2 0.2aB 0.30 0.2aA 12.1 0.3aA
25 mg PCP L1
S. brumptii aA
85.0 0.0 7.7 0.1aB 7.2 0.9bA 126.5 2.1bB 106.33 8.0bB 0.0 0.0aB 8.3 0.1bB 0.60 0.2aA 11.8 0.2aA
B. nivea
S. brumptii aB
64.8 17.5 5.8 1.5aC 3.2 0.9aB 58.1 4.2aB 50.27 5.0aB 0.0 0.0aB 6.1 0.1aC 1.10 0.4aB 23.8 0.4aB
83.3 2.5bA 7.5 0.2bB 7.2 0.7bA 43.3 1.4bC 27.7 7.0bC 0.0 0.0aB 4.3 0.1aC 0.80 0.2aA 24.1 0.2aB
Different lower case letters (a and b) refer to significant differences (P < 0.05) between PCP sensitivity, adsorption and degradation of B. nivea and S. brumptii at the same PCP concentration. Different upper case letters (A, B and C) refer to significant differences (P < 0.05) among PCP sensitivity, adsorption and degradation of B. nivea and S. brumptii at different PCP concentration.
fungi in liquid culture (Table 1). In the control, without fungal mycelium, the PCP removed was negligible.
Electrolyte Leakage Assay
and 3 y, respectively. Finally, only 60 % of plants used for control were found alive after 1 y, while, all chestnut plants died after 3 y in treatments with only the pathogens without the antagonist (Table 2). P. cambivora and P. cinnamomi were the
The conductance of all samples increased over time reaching a stable value after 7 h (Fig 1). The incubation of Byssochlamys nivea and Scopulariopsis brumptii with PCP did not produce any PCP toxic effects on mycelium without significant variation of electrolyte leakage after 24 h (Fig 1a,b). The experiment carried out on vegetable tissues highlighted that the conductance of all systems increased by about 80 % over time (Fig 2) and no significant effect of fungal metabolites on tomato stems was ascertained at 24 h. However, an increase of electrolyte leakage of the tomato stem was registered during the first eight hours in the presence of B. nivea and in the range of 2e5 h in the presence of S. brumtii respect to control.
Antagonistic assays of Byssochlamys nivea and Scopulariopsis brumptii versus Phytophthora cambivora and Phytophthora cinnamomi under laboratory and greenhouse conditions Radial mycelium extension was measured on PDA in colony interactions and VOC experiments. In the first case, the growth of Phytophthora cambivora and Phytophthora cinnamomi was reduced to 40 % and 60 % when these pathogens were cultured with Scopulariopsis brumptii and Byssochlamys nivea, respectively (Table 2). The radial growth of Phytophthora isolates, measured in VOC experiments in presence of the antagonist fungi, showed no significant difference respect to the control (Table 2). Finally, S. brumptii and B. nivea reduced to 50 % and 60 % the fungal mat production of P. cambivora and P. cinnamomi in flask (Table 2). In greenhouse experiments, the survival of chestnut plants was recorded annually for three years. The highest percentage of plants survived was found in the treatment with S. brumptii (about 70 %) against both P. cambivora and P. cinnamomi until the end of the experiment (Table 2). The plants survived in the treatment with B. nivea were about 70 % and 50 % after 1
Fig 1 e Conductance values (mean ± standard deviation) during 24 h of incubation using Electrolyte Leakage Assay (ELA) of: a) B. nivea incubated in distilled water (white bar) and B. nivea incubated at 25 mg PCP LL1 (black bar); b) S. brumptii incubated in distilled water (light grey bar) and S. brumptii incubated at 25 mg PCP LL1 (dark grey bar). Different small letters (a and b) refer to significant differences (P < 0.05) among PCP sensitivity. The conductance values were calculated as the difference from the reading at the beginning of the assay (increments in microsiemens from t0).
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
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L. Bosso et al.
Fig 2 e Conductance values (mean ± standard deviation) of tomato stems in the presence of B. nivea (black bars) and S. brumptii (grey bars) culture filtrates during 24 h of incubation using Electrolyte Leakage Assay (ELA). Different small letters (a, b and c) refer to significant differences (P < 0.05) among treatments at the same time. The conductance values were calculated as the difference from the reading at the beginning of the assay (increments in microsiemens from t0).
cause of dead chestnut plants in accordance with Koch’s postulates.
Discussion Byssochlamys nivea and Scopulariopsis brumptii in PCP sensitivity The fungal strains showed a good PCP sensitivity at the concentrations tested. In Petri dishes, Byssochlamys nivea suffered a decrease of radial mycelium extension, hyphal thickness and daily mycelium growth rate in the presence of 25 mg PCP L1. Instead, Scopulariopsis brumptii grew without significant limitations due to PCP. A strain of Rhizopus nigricans was able to grow even at 100 mg PCP L1 while no growth was detected at 250 mg PCP L1 (Tomasini et al. 2001). Moreover the R. nigricans mycelium daily growth rate was 9.8 and 9.6 mm day1 with 12.5 and 25 mg PCP L1, respectively. Our fungi were slower in growth (in average 7 mm day1 for both
fungi) than R. nigricans, but, after 5 d, the radial mycelium extension of our fungi was 55 mm against to 45 mm of R. nigricans. The radial growth of Aspergillus niger was completely inhibited at 40 mg PCP L1, while at concentrations of 10 and 20 mg PCP L1, the mycelium growth was reduced to 75 % and 88 %, respectively (Bomar & Bomar 1999). At the same PCP concentrations, A. niger formed only vegetative structures without conidia formation. B. nivea and S. brumptii did not suffer of the same radial extension reduction, although, the spore production decreased by increasing PCP concentration. After the experiment the conidia were still able to germinate for both fungi. Phanerochaete chrysosporium showed a daily mycelium growth rate of 10.3 cm day1 at 12.5 mg PCP L1, but its growth rate decreased until 8.2 cm day1 by increasing PCP concentration at 25 mg PCP L1 (Tomasini et al. 1996). Respect to P. chrysosporium, our fungal strains showed the same daily mycelium growth rate at 12.5 mg PCP L1, while they were slower than P. chrysosporium at 25 mg PCP L1. White-rot Chilean isolates were tested in resistance and mycelium radial extension at 25 mg PCP L1 in N-limited solid media (Tortella
Table 2 e Antagonistic assays of B. nivea and S. brumptii against P. cambivora and P. cinnamomi under laboratory and greenhouse conditions. Without fungal antagonist P. cambivora Colony interaction (mm) Volatile organic compounds (mm) Fungal mat (mg) Chestnut live plants after 1 ya (%) Chestnut live plants after 2 ya (%) Chestnut live plants after 3 ya (%)
aA
42.5 0.5 42.5 0.5aA 8.7 0.5aA 60 10 0
P. cinnamomi bA
40.1 0.2 40.1 0.2bA 8.2 0.5aA 60 10 0
B. nivea P. cambivora aB
12.3 0.2 41.3 0.8aA 2.9 0.5aB 72 60 50
S. brumptii
P. cinnamomi aB
12.8 0.5 40.1 0.6aA 3.0 0.2aB 65 65 50
P. cambivora aC
24.2 0.6 41.3 0.8aA 5.2 0.5aC 75 70 70
P. cinnamomi 23.2 0.8aC 40.3 0.6aA 5.6 0.3aC 75 75 70
Different lower case letters (a and b) refer to significant differences (P < 0.05) between growth values of P cambivora and P. cinnamomi versus the same fungal antagonist. Different upper case letters (A, B and C) refer to significant differences (P < 0.05) among growth values of P cambivora and P. cinnamomi versus different fungal antagonist. a Greenhouse experiments.
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
B. nivea and S. brumptii like PCP degraders and fungal antagonists
et al. 2008). When the fungi were exposed to PCP, only Lenzites betulina had a growth rate of 10 mm day1 similarly to our fungi tested in this study. Patulin and fungal mat production were negatively influenced by increasing PCP concentration. In literature there are no studies on the interaction between the patulin production and PCP. B. nivea and S. brumptii reduced by 60 % the patulin production at 25 mg PCP L1. Same results were obtained when the fungi were grown in PDB to evaluate the fungal mat. Fungal strains were cultured for 7 d with 25 mg PCP1 in liquid batch and they produced a reduction in growth of the fungal mat by 74 % in Volvariella volvacea; 77 % in Armillaria gallica; 30 % in Armillaria mellea; 28 % in Ganoderma lucidum and 17 % in Pleurotus pulmonarius (Chiu et al. 1998). In cultures with 25 and 12.5 mg PCP L1, the fungal mat growth of Amylomyces rouxii was 8 mg after 96 h (Marcial et al. 2006). In latter case the fungal mat growth was significantly lower than our fungi. When B. nivea and S. brumptii were cultured in liquid mineral medium with PCP as sole carbon source, the fungi showed no fungal mat production. Instead when glucose was added in the flask, fungal mat was achieved for both fungi. This essentially happened because, unlike bacteria, fungi do not utilize PCP as a source of carbon and energy (McAllister et al. 1996). In fungi the degradation of PCP is not consequence of enzyme tools which are able to do this function but an effect of cometabolism (McAllister et al. 1996; Bosso & Cristinzio 2014). The effects of PCP on hyphal size, mycelium morphology and spore production of B. nivea and S. brumptii were made more evident by increasing PCP concentration, although the PCP never reduced u the fungal growth of B. nivea and S. brumptii to zero. ELA assays showed that B. nivea and S. brumptii, after 24 h of incubation at 25 mg PCP L1, did not suffer a decrease in electrolyte leakage compared to control incubated in distilled water. It is widely known that PCP can negatively influence fungi cellular processes, morphology, lipid membrane components, fungal mat growth, enzymatic activity, sporulation and reproduction capacity (Watanabe 1978; Bajpaia & Banerjib 1992) but, in our case, B. nivea and S. brumptii seem to have a good sensitivity to PCP. Sensitivity to PCP represents the most important characteristic required to select specific and effective microorganisms useful in PCP degradation.
Efficiency of Byssochlamys nivea and Scopulariopsis brumptii in PCP removal Fresh mycelium of Byssochlamys nivea and Scopulariopsis brumptii was able to adsorb and degrade PCP. While for both fungi the adsorption was <5 %, B. nivea and S. brumptii degraded about 95 % of the PCP after 28 d of incubation (Table 1). These fungal strains are known to not produce extracellular enzymes as lignin peroxidases or laccase which are highly efficient in degrading PCP in co-metabolism (McAllister et al. 1996; Bosso & Cristinzio 2014). However, S. brumptii produce phenoloxidase enzymes (Tanaka et al. 2000) which can be useful in PCP degradation (Bosso et al. 2015b). B. nivea was able to degrade 12.5 and 25 mg PCP L1 after 28 d in flask due to the pectinolytic enzymes, which are widely used in bioremediation strategy (Chiu & Chang 1973; Bosso et al. 2011; Bosso et al. 2015b). In fact, in Byssochlamys genus
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some enzymes were detected useful in the degradation pathway of lignin and other wood constituents (Chiu & Chang 1973: Furukawa et al. 1999). The ability of B. nivea and S. brumptii to remove PCP was excellent after 28 d of incubation. A strain of Byssochlamys fulva was able to remove 20 % of 50 mg PCP L1 in only 8 d (Scelza et al. 2008). Excellent results in PCP depletion were also obtained with an isolate of Rhizopus nigricans (Tomasini et al. 2001). In fact the strain degraded 12.5 mg PCP L1 using phenoloxidase activity in only 8 d. In N-limited liquid medium Anthracophyllum discolor, Lenzites betulina and Galerina patagonica removed 25 mg PCP L1 in only 15 d thanks to manganese and lignin peroxidase production (Tortella et al. 2008). The maximum efficiency in PCP removal was obtained by Ganoderma lucidum, Phanerochaete chrysosporium and Polyporus sp. after 7 d of incubation in liquid batch system with 25 mg PCP L1. These fungi were able to remove 75e78 % of the PCP initial concentration (Chiu et al. 1998). The majority of fungi implicated in PCP degradation are members of the white-rot Basidiomycetes and are capable of degrading lignin. Almost all studies on PCP degradation were conducted with P. chrysosporium, Phanerochaete sordida, Trametes versicolor and Trametes hirsuta (McAllister et al. 1996; Bosso & Cristinzio 2014). Few studies about PCP removal by Ascomycota fungi are available in literature (Rubilar et al. 2012; Bosso et al. 2015a). This justifies the importance to study fungi belonging to this fungal group that may tolerate and degrade high PCP concentrations.
Biocontrol of Phytophthora cambivora and Phytophthora cinnamomi by Byssochlamys nivea and Scopulariopsis brumptii Byssochlamys nivea and Scopulariopsis brumptii have demonstrated excellent capacity to reduce the growth of Phytophthora cambivora and Phytophthora cinnamomi in all microbiological media. Several antagonists and their metabolites were found useful in Phytophthora’s control. A Flufluran derivate extract by Aspergillus flavus completely inhibited the mycelium growth of P. cinnamomi after 7 d (Evidente et al. 2009). Antibiosis and mycoparasitism action by some fungi and bacteria, isolated by manure compost, were able to lyse Phytophthora’s mycelium with inhibition values between 40 and 70 %. b-glucosidase and phosphates activities of a Trichoderma sp. strain reduced the development of P. cinnamomi (Kelley & Rodriquez-Kabana 1976). A methanolic compound obtained from the cyanobacterium Nostoc was tested with good results against a variety of pathogens of agricultural importance among which: P. cambivora and P. cinnamomi (Aryantha & Guest 2006). Antibiosis between our fungi and Phytophthorae was due essentially to the patulin. Patulin is a mycotoxin with antagonist capacity against a wide spectrum of microorganisms. Patulin produced by Aspergillus clavatonaticus exhibited inhibitory activity in vitro against several plant pathogenic fungi i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum, Rhizoctonia solani and Pythium ultimum (Zhang et al. 2008). This mycotoxin is able to stop the synthesis of rRNA, tRNA and mRNA in Saccharomyces cerevisiae (Sumbu et al. 1983) and to have antibacterial activities against Escherichia coli and Micrococcus luteus (Praveena & Padmini 2011). P. cambivora and P. cinnamomi showed no significant decrease in radial growth when cultured in VOC experiments.
Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004
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The VOCs are often used in Phytophthora control but our experiments not appear effective to limit the growth of the pathogens. The endophytic fungus Muscodor crispans produced VOCs that have inhibitory effects against P. cinnamomi, P cambivora and other oomycetes such as Phytophthora palmivora, P. ultimum (Mitchell et al. 2010). Furthermore Nodulisporium sp., another endophytic fungus, produces VOCs inhibitors against a number of pathogens such as Aspergillus fumigatus and R. solani, P. cinnamomi and Sclerotinia sclerotiorum within 48 h of exposure (Mends et al. 2012). The potential of B. nivea and S. brumptii as biocontrol agents to suppress P. cinnamomi and P. cambivora was evaluated in greenhouse experiments. B. nivea and S. brumptii had a biocontrol on Phytophthora species. No chestnut plants survived in the control after 3 y. On the other hand, we obtained a survival of chestnut plants >50 % in the treatments with B. nivea and S. brumptii (Table 2). Several microorganism antagonists to P. cambivora and P. cinnamomi have been described in literature, providing considerable scientific interest, as well as: Trichoderma harzianum, Bacillus subtilis, Penicillium funiculosum and Gliocladium virens (Howell 2003; Sylvia et al. 2005; Griffin 2014). Although antibiosis seems to be the strategy involved in the biocontrol activity by B. nivea and S. brumptii, further analysis of their modes of action are currently being undertaken in order to improve their use and implementation under real culture conditions. Biocontrol by B. nivea and S. brumptii of P. cambivora and P. cinnamomi seems to be a good alternative for sustainable agriculture to overcome the problems of public concern associated with pesticides and pathogens resistant to chemical pesticides. Ink disease, caused by the oomycete pathogens P. cambivora and P. cinnamomi, is probably the most prevalent Chestnut disease. It is very destructive and causes flame shaped dark necroses on collar rot of adult trees, shrubs and seedlings. Biological control is an excellent alternative for sustainable agriculture to avoid problems associated with the use of pesticides. Many studies have been done to date; although almost always conducted at laboratory scale. In this regard, B. nivea and S. brumptii do not produce toxic compounds able to damage cell walls and membranes of tomato stem cells; as shown in the ELA experiment (Fig 2). In contrast, the incubation of tomato stems in A. flavus culture filtrates produced high phytotoxicity due probably to hydrophilic metabolites that remainin the aqueous phase (Evidente et al. 2009). In our experiments no significant difference in conductance was detected between the tomato stem incubated in distilled water and in B. nivea or S. brumptii culture filtrates. The next research step is to address in a field study the results obtained in this research.
Conclusions Critical analysis reveals that there are relatively few reports on the use of microorganisms in the double role of bioremediation and biocontrol agent. This is the first paper about the capacity of Byssochlamys nivea and Scopulariopsis brumptii to remove PCP and to control the Phytophthora species agents of Ink disease on chestnut. Furthermore, the excellent result showed by ELA assays on tomato stems indicate that our fungi do not cause significant damage to plants and that they can be
L. Bosso et al.
used in field experiments. For this reason, our results indicate that B. nivea and S. brumptii have an interesting potential to be used in bioremediation and biocontrol strategy.
Acknowledgements We thank Prof Dianna Pickens for the linguistic revision.
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Please cite this article in press as: Bosso L, et al., Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.01.004