Basidiomycetes as Potential Biocontrol Agents against ... · entomophagous and nematophagous specialized fungi (YANG& al. [9]). These organisms have the advantage of being effective

Romanian Biotechnological Letters Copyright © 2016 University of Bucharest Vol. 21, No. 1, 2016 Printed in Romania. All rights reserved ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 21, No. 1, 2016 11185

Basidiomycetes as Potential Biocontrol Agents against Nematodes

Received for publication, June 12, 2015 Accepted, December 20, 2015

TIBERIUS BALAEȘ 1*, CĂTĂLIN TĂNASE2

1Anastasie Fatu Botanical Garden, AlexandruIoanCuza University of Iasi, Iasi, Romania Dumbrava Rosie Street, No. 7-9, 700487 Iasi, Romania;

2Department of Biology, Faculty of Biology, Alexandru Ioan Cuza University of Iasi, Bd. Carol I, No. 20 A, 700505 Iasi, Romania

*Corresponding author: tel: 00400749186167; e-mail: [email protected]

Abstract The study aims to investigate the potential of some basidiomycete species to be used as agents for

biocontrol of nematodes, a subject very little exploited. Saprophytic fungi degrade dead organic substrates, but can colonize living organisms in order to increase the survival chances in conditions of low nutrient availability. In the framework of this study, 67 species of basidiomycetes were tested concerning their potential of using nematodes as sources of nutrients. The mycelium of basidiomycete species was tested on solid nutritive media to assess their efficiency in colonising nematode’s bodies. We have evaluated various carbon and nitrogen sources in terms of quality and quantity in order to optimize the culture conditions. Macroscopic and microscopic analysis showed partial or total fungal colonization of nematode’s bodies for 35 out of 68 fungal species. Hyphae developed in or on the nematode’s bodies were observed, causing degradation of cuticle. Further analysis revealed potential for using nematodes as nutrients for 3 species of basidiomycetes and also the production of some nematicidal compounds. The most important factors affecting the process were the quality and quantity of nutrients. These results have implication in the possible use of fungi in biocontrol of telluric phytopathogens.

Keywords: biocontrol, adaptive metabolism, nematodes, nematophagous fungi

1. IntroductionSaprotrophic fungi degrade dead organic substrates, but can attack living organisms for

the use of alternative resources to increase the chances of survival under conditions of low nutrient availability (DIX and WEBSTER [1]). These adaptive organisms are of particular interest due to the possibility of being used as biological control agents of phytopathogens, in the frame of the increased worries regarding side effects from the use of chemicals such as: environmental pollution, contamination of agricultural products, increased risk on human health and aquatic organisms, non-specific effect on beneficial soil biota and development of pathogen resistance to the action of pesticides (DENT& al.[2]).Saprotrophic basidiomycetes that decay wood or litter, present an increased adaptability to the substrate and also the ability to use a wide range of nutrients. The property of saprotrophic basidiomycetes to grow in different soil types provides a major advantage over obligated entomophagous or nematophagous fungal species due to the possibility of being applied as inoculum in the soil of interest and to resist / develop for an extended period. Moreover, high concentrations of

TIBERIUS BALAEȘ, CĂTĂLIN TĂNASE


heavy metals in soil inhibit the growth of entomophagous fungi (GRAY [3]).The biopesticidal activity of fungi was revealed for a wide range of pathogens: mites (MONTEIRO& al. [4]), insects (ALI & al. [5]; BAVERSTOCK & al. [6]), nematodes (REGAIEG & al. [7]; GINÉ & al. [8]) etc. As main strategies are notable: parasitism, toxicity and active capture. To date, most studies on biological control using fungi were oriented toward regular imperfect fungi or entomophagous and nematophagous specialized fungi (YANG& al. [9]). These organisms have the advantage of being effective in infecting and destroying the host, but the increased dependence of the host presence gives the disadvantage of not efficiently develop in the ground and to be difficulty produced at industrial scale. Some formulations (biopesticides) have been commercialization (LEATHERS [10]).Although less studied, basidiomycetes can be very effective in "capturing" and consuming invertebrates. The species from genus Pleurotus have been studied extensively in this regard: Pleurotus cystidiosus presents toxocysts, surrounded by a droplet of toxin that paralyzes nematodes (TRUONG& al. [11]); Pleurotus ferulae produces compounds with nematicidal activity (LI & al. [12]); Pleurotus ostreatus capture 30-50% of the nematodes that come in contact with the fungal hyphae (TRUONG & al. [11]) and also numerous insects larvae (DIX and WEBSTER [1]). Among the species of basidiomycetes consuming invertebrates there are included: Coprinuscomatus (LUO& al. [13]), Hohenbuehelia (HIBETT and THORN [14]; KUMAR and KAVIYARASAN [15]), Hyphoderma spp. (TZEAN and LIOU [16]), Stropharia rugosoannulata (LUO & al. [17]). The mechanisms involve the production of specialized structures - acanthocysts, stephanocysts and echinocysts (KARASIŃSKI [18]) that function as attractants and for the cuticle penetration, or contain paralyzing toxins. Many species of basidiomycetes produce compounds with nematicidal activity – Coprinus xathothrix (LIU & al. [19]), while the culture filtrate of many basidiomycetes (Lentinula, Oudemansiella, Peziza, Pleurotus, Daedalea, Fistulina, Ramaria, Tylopilus) has nematotoxic activity (DONG and ZHANG [20]). The total number of biopesticide isolated from basidiomycetes is very high (LORENZEN and ANKE [21]). In this study, a selection of basidiomycete strains that use nematodes as sources of nutrients is presented. The possible mechanisms involved in the process is discussed. The strains of Daedalea quercina, Fomitopsis pinicola and Gymnopilus junonius proved a strong efficiency in destroying and colonising nematodes.The present results offer a new perspective concerning the nutritional adaptations of some basidiomycetes, with possible implications in the use of these organisms as biocontrol agents of soil-born phytopathogenic nematodes!

2. Materials and methods

a. Fungal strains and inocula All the tested fungi are from the Culture Collection of Fungal Research Laboratory

(RECOSOL), Faculty of Biology, Alexandru Ioan Cuza University of Iasi, previously isolated using fruit bodies collected from wild habitats (BALAEȘ and TĂNASE [22, 23]; PETRE and TĂNASE [24, 25]). An organic medium was used for preparation the inocula (L-1): glucose 8 g, malt extract 20 g, yeast extract 2 g, peptone 2 g, agar 15 g.

b. Nematodes The nematode species – Steinernema carpocapsae was purchased as a commercial

available product, Capsanem® (Koppert B.V, The Netherlands) consisting of a powder with juveniles of approximately same stage of development.

c. Media and cultures The preparation of all the culture media has been realised using reagents of analytical



grade, acquired from Merck & Co., Inc. and Carl Roth GmbH + Co, KG. Eight types of organic media were tested for analysing the potential of fungal species to use nematodes as sources of nutrients, the composition being given in the Table 1. No other minerals have been added, except the hydrochloric acid 0.1 M or potassium hydroxide 0.1 M in order to adjust the pH at the value 5.5 prior to sterilisation. All the media were autoclaved at 120 ºC. Three replicates have been made for all the samples. The pH was adjusted using an electronic pH/ion-meter (model INOLAB, WTW, Weilheim, Germany). The plates have been incubated in the dark at 25 ºC, for 10 days before nematodes being added.

The inoculation of mycelium was performed using agar plugs of 11 mm diameter, taken from colonies in active growth stage (2 weeks old cultures, grown on the inoculum medium, as described above) and placed in the centre of each Petri plate. After 10 days of incubation, 1 mL of nematodes suspension (1000 nematodes) has been pulverized on the surface of each Petri plate, being analysed for 40 days further. At this time, the fungal colonies were 0.5-5 cm in radius, depending on the media type. Controls, consisting of Petri plates inoculated with nematodes but with no fungi, were used for each type of media. After selecting the most efficient fungal species, cultures supernatant was tested for nematicidal activity.

Table 1. The composition of nutritive media (in g L-1)

Media Agar Glucose Peptone Yeast extract Malt extract I 15 10 3.5 3.5 3.5 II 15 10 0.8 0.8 0.8 III 15 10 0.7 0.7 1 IV 15 10 - - - V 15 2 0.5 - - VI 15 - - - - VII 15 1 0.5 - - VIII 15 2 - - -

The medium V (2g L-1 glucose, 0.5g L-1 peptone) was also used for assessing the

influence of pH on the mortality of nematodes under fungal cultures for three of the fungal species. The pH values were: 4.0, 5.0, 6.0, 7.0 and 8.0.

d. Analytical methods and measurements All the cultures were analysed starting 10 days after nematode suspension was added, for

40 days. In this respect, visual observation under the stereomicroscope (stereomicroscope SZM2 Optika, at a magnification of 20-45x) were made, and the viability of nematodes was assessed based on mechanical stimulation with a very thin needle. The nematode’s bodies colonisation by fungal mycelium was assessed visually, and nematodes at all stages of fungal colonisation have been analysed under microscope (phase contrast microscope NIKON, at a magnification of 200-1000x) after staining. Pictures have been taken using a FujiFilm AV200 photo camera. SEM analysis hasbeen performed on a Quanta 250, FEI SEM, in high vacuum mode, the probe being dehydrated at room’s temperature prior to analysis.

e. Statistical analysis In order to evaluate the rate of fungal colonisation on the nematode’s bodies, and

viability of nematodes, on each Petri plate, 7 squares of 1 cm2 were randomly chosen, and in the designated surface all the nematodes were counted, either dead or alive, colonised or non-colonised. For each plate 210-250 nematodes were counted. All the data were normalised and the mean value for each data set (7 squares X 3 plates / species / medium) was calculated and the obtained value was considered the percentage of mortality / colonisation.



3. Results and discussion The results obtained have shown a good activity in colonising nematodes for some of the

tested strains. All the fungal strains were tested on the first three types of media, rich in nutrients. No colonisation of nematode’s cadavers was observed on these media, suggesting that when adequate nutrients are available for the mycelium, there are no changes in the metabolism and the nutrition strategy of the fungi, as other researchers also found (ANANKO and TEPLYAKOVA [26]). On a medium without nitrogen sources (medium IV), some of the strains adapted their metabolism and nutrition for using nematodes as a source of nutrients. This phenomenon have been highlighted through visual observation (both on stereomicroscope and microscope as well), nematode’s bodies being colonised by the fungal mycelium (Fig. 1(a) and 1(b)). From this colonised cadavers a cluster of hyphae have been developing (Fig. 1(c) and 1(d)), with the production of abundant chlamydospores near nematode’s cadavers, proving that these organisms represented a good source of nutrients.

Fig. 1. Colonisation of nematode’s bodies – initial stages of infection (a andb) and the end of colonisation and degradation (c and d). The arrows indicate the point of infection.

At the end of the process, the nematode’s body is completely colonised with hyphae

developing inside it and the cuticle being degraded. As shown in Fig. 2, hyphae form networks inside the body exploiting the nutrients found here, and are developing in dens clusters comparing to the medium.

Fig. 2. Scanning electron microscopy photographs – un-colonised (a) and colonised (b–d) nematodes (the arrows indicate the hyphae emerging from the body)

From the 68 strains tested, 33 did not colonise the nematode’s bodies, while 7 strains

have poorly colonised them after a long period of time (30-35 days), when cadavers were starting to disintegrate. However, 28 strains colonised well the nematodes and have developed hyphae and completely degraded these bodies. The most efficient 22 strains were selected for further studies on medium V and medium VI. Due to the lower content of glucose, a slow rate of fungal development has been observed (plates were covered with a very lax mycelium in more than 2 weeks), and some of the strains did not develop at all. Based on mortality of



nematodes and the rate of fungal colonisation of them per unit of time, five classes of efficiency have been created for selecting the most promising strains (Table 2).

Table 2. The anti-nematode activity of 22 of the strains on media V and VI, after 32 days of incubation (the numbers represents the classes of efficiency: 0 – the absence of activity, 1 – very slow activity,

2 – reduced activity, 3 – good activity, 4 – very good activity) Species Medium Species Medium

V VI V VI Bjerkandera adusta 1 1 Laetiporus sulphureus 0 0Crepidotus applanatus 0 0 Megacollybia platyphylla 4 0 Cyathus striatus 2 1 Meripilus giganteus 1 0 Daedalea quercina 4 0 Peniophora quercina 0 0 Daedaleopsis tricolor 1 0 Phlebia radiata 0 0 Fomitopsis pinicola 4 0 Pleurotus ostreatus 3 0 Ganoderma applanatum 1 1 Schizophyllum commune 2 1Gymnopilus junonius 4 2 Trametes ochracea 0 0 Hymenopellis radicata 0 0 Trametes suaveolens 0 0 Irpex lacteus 1 0 Trametes trogii 1 0 Kuehneromyces mutabilis 1 1 Tricholomas ejunctum 0 0

The colonisation started from a point at the nematode’s surface (Fig. 3(a) and 3(b))

where hyphae were developing in a cluster or by surrounding the body with specialised hyphae (Fig. 3(c) and 3(d)). A development of hyphae in the entire body could be observed, followed by an advanced degradation (both the cuticle and the content inside) of the body (Fig. 3(e) and 3(h)). Hyphae were developing in much denser cluster where bodies were present, forming abundant chlamydospores or conidia.

Fig. 3. Different stages of colonisation and degradation of nematode’s bodies: a–d – initial stages and e–h – the end of the process(a-d and f-h – 400x magnification; e – 100x magnification;

a, b, e and h – phase contrast; (c, d and g – micrometre eye piece, 1 major unit correspond to 25 µM) Differences have been observed among the fungal species tested concerning the way the

infection and later the hyphae’s development are evolving. Increasing rate of mortality and colonisation was different, being approximately simultaneous for Cyathus striatus, Gymnopilus junonius and Kuehneromyces mutabilis, and suggesting a biological mechanism of action, while for the other species colonisation rate was delayed (data not shown), and a



chemical mechanism is believed to occur. This phenomenon was particularly observed for Fomitopsis pinicola and Daedalea quercina. The production of nematicidal compounds by F. pinicola has been reported previously (CHEN & al. [27]). In the case of G. junonius both a biological and a chemical mechanisms might be involved. The production of antimicrobial compounds by G. junonius has been reported (AQUEVEQUE & al. [28]) and also the production of Gymnopilin, a substance with effect over the mammalian nervous cells (MIYAZAKI& al. [29]). Other species in this family produce “biological weapons” to attack nematodes (LUO& al. [17]).S. TZEAN and J. LIOU [16] suggested that these structures might be produced by many species of basidiomycetes. The most efficient strains were D. quercina, F. pinicola,G. junonius, M. platiphylla and P. ostreatus.

While it is a known strategy for P. ostreatus to colonise and consume insect larvae and nematodes (DIX and WEBSTER [1]; TRUONG& al. [11]) the other species are less studied for these properties, this information being a novelty in the field. Although confirmed from different authors as a species that actively capture nematodes (HEYDARI & al. [30]), P. ostreatus did not prove the highest efficiency in our study.

In Fig. 4 there is presented the viability of nematodes on media V-VIII inoculated with the five species and C. striatus, through a statistical approach (choosing randomly sites of 1 cm2). Percentages of nematode’s mortality were calculated, in comparison with controls (fungi free). It can be observed that the principal factor that influenced the colonisation rate was the quantity of glucose, 2 g of glucose L-1being sufficient for mycelium to develop and to switch in the direction of metabolizing nutrients from nematode’s bodies. This suggests that the availability of sugars is a very important factor in mycelium’s development, being necessary an adequate quantity of sugars for the hyphae to emerge. The addition of 0.5 g peptone is slowly stimulating the colonisation process, but colonisation occurs also in the absence of it. The wood decaying fungi are adapted for substrates with a low content in nitrogen, with a C: N ratio very high (BARRON [31]).

Fig. 4. The efficiency of fungal colonisation on nematodes’ bodies on different media: V, VI, VII and VIII (C – control, CS – Cyathus striatus, DQ – Daedalea quercina, FP – Fomitopsis pinicola,

GJ – Gymnopilus junnius, MP – Megacollybia platyphylla, PO – Pleurotus ostreatus) When nitrogen is available in very low amounts, many species of basidiomycetes are

using organic debris from soils, feeding on bacteria (BARRON [31]) or capturing and



decomposing invertebrates. Through these strategies, basidiomycetes are satisfying their nitrogen requirements, but the lack of sugars remain a limiting factor. J. XUE and co-workers [32] showed that a nematophagous specialised fungus Esteya vermicola require a substantial amount of sugars to produce abundantly conidia that adhere to nematodes. The most efficient strains were D. quercina, F. pinicola and G. junonius, which finally colonised all the nematodes, although on the control plates the nematode’s viability was still very high. These species were further used for assessing the influence of pH, on the medium V (Table 3), for values of pH from 4.0 to 8.0. The obtained results prove that F. Pinicola prefers an acidic medium (pH 4.0), while G. junonius and D. quercina prefer a moderate acidic medium.

Table 3. Mortality of nematodes on media with different values of pH

(composition of media L-1: 2 g glucose, 0.5 g peptone) pH D. quercina F. pinicola G. junonius Control 4 38% 51% 20% 15% 5 39% 36% 50% 14% 6 50% 20% 34% 10% 7 37% 16% 15% 5% 8 35% 15% 10% 5%

On the same medium (V), the three species of fungi where co-inoculated with nematodes

on compartmented Petri dishes that have a septum dividing the plate in two. Half of each plate was inoculated with fungi, while the other half was inoculated with nematodes. The difference in nematode’s mortality on the plates with D. quercinaand F. pinicola was not significant, while on the plate with G. junonius the mortality reached 100% compared with the 3% on the control plate. Volatile compounds with nematicidal activity might be produced by this fungal species. The mechanisms involved in increasing mortality of nematodes can be either of biological or biochemical type. In this respect, the supernatant obtained from cultures of the three species was incubated with nematodes for 24 hours (Table 4). Fungal cultures free of nematodes and cultures of fungi inoculated with nematodes for an induced response were both used in this experiment. Control consisting in fungi-free liquid media were also used. The results showed a very strong nematicidal activity of the all types of supernatant, the strongest effect being produced on nutrients-reach media, probably due the very fast development of fungal mycelium and the production of secondary metabolites.

Table 4. Mortality of nematodes on supernatant obtained from 7 days old cultures

(after 24 hours of incubation, DQ – D. quercina, FP – F. pinicola, GJ – G. junonius, GJN – G. junonius + nematodes)

Control DQ FP GJ GJN Composition of media (L-1)

5% 9% 28% 55% 45% 3 gglucose, 0.5 g peptone, 1 g KH2PO4; 0,5 MgSO4

.7H2O; 0,1 FeSO4

.7H20; 0,1 NaNO3

5% 11% 38% 88% 76% 5 gglucose, 1 g peptone, 1 g KH2PO4; 0,5 MgSO4

.7H2O; 0,1 FeSO4

.7H20; 0,1 NaNO3 3% 94% 99% 100% 100% 10 gglucose, 5 g peptone, 1 g malt extract, 1g yeast extract

The fungi, and particularly the wood decaying basidiomycetes, can recycle their own

nutrients from old degenerated hyphae in order to extend the mycelium in searching for new resources (DIXand WEBSTER [1]). This property is very important, especially on agricultural soils that have often a low content in organic compounds. This might be an advantage comparing to the nematophagous specialised fungi that are more sensitive to soil’s properties



(GRAY [3]). Although in the soil there are usually smaller amounts of sugars necessary for development of ligninolytic fungi, here the mycelium can use a variety of other organic nutrients, and even more, in the rhizosphere soils, the mycelium can use sugars excreted by the roots. This is particularly important since the phytopathogenic nematodes attack the roots.

4. CONCLUSIONS

In summary, many species of basidiomycetes, especially the wood decaying ones have a versatile metabolism that allow a switching of nutrition strategy in stress conditions, from degrading the specific substrates (e.g. wood, litter) to degrading various other substrates and using them as sources of nutrients. Among the species of basidiomycetes that can use cadavers of invertebrates as a substrate, there are some that actively capture these organism through chemical or biological mechanisms. These properties of basidiomycete species confers the possibility of using them in strategies of biocontrol of soil-born phytopathogenic invertebrates, particularly phytopathogenic nematodes. In our study, the strains of Daedaleaquercina, Fomitopsispinicola and Gymnopilusjunonius proved to be highly effective in colonising nematodes and with a strong nematicidal activity. Their efficiency was dependant on the nutrient medium composition, decreasing when nutrients were in large quantity. A small concentration of glucose is necessary for the process to occur. Additional studies are required in order to optimise the process.

Acknowledgements. This work was supported by the strategic grant POSDRU/159/

1.5/S/133391, Project “Doctoral and Post-doctoral programs of excellence for highly qualified human resources training for research in the field of Life sciences, Environment and Earth Science” co-financed by the European Social fund within the Sectorial Operational Program Human Resources Development 2007–2013”. The POSCCE-O 2.2.1, SMIS-CSNR 13984-901, No. 257/28.09.2010 Project, CERNESIM, is gratefully acknowledged for the infrastructure (SEM) used in this work.

The authors declare that they have no conflict of interest.

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Basidiomycetes as Potential Biocontrol Agents against ... · entomophagous and nematophagous specialized fungi (YANG& al. [9]). These organisms have the advantage of being effective