Potential of Some Nematophagous Fungi against Meloidogyne hapla Infection in Czech Republic

Manzoor Hussain*, Miloslav Zouhar and Pavel Ryšánek

Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Prague, Czech Republic

ABSTRACT

Five fungal species Arthrobotrys oligospora, Dactylella oviparasitica, Clonostachys rosea, Stropharia rugosoannulata and Lecanicillium muscarium isolated from root and soil samples collected in Prague, Czech Republic, were cultured on agar media and tested against Meloidogyne hapla both in-vitro and in-vivo. All fungi proved to be efficient in reducing final population of northern root knot nematode M. hapla and giving vigour to the plants. In laboratory experiment, L. muscarium was the most effective against nematode eggs (95.6%) and second stage juveniles (J2) (95.8%) infection. In greenhouse experiment, similar trend was found. L. muscarium proved to be more effective against M. hapla whereas S. rugosoannulata and C. rosea showed better results among other tested fungi in experiments. Moreover, plant growth parameters were also improved due to antagonistic relationship of fungi and nematodes in soil.

Article Information

Received 2 June 2016

Revised 29 June 2016

Accepted 16 July 2016

Available online 01 October 2016

Authors’ Contributions

MH designed study, collected data and wrote manuscript. MZ and PR supervised the study and proofread manuscript.

Key words

Meloidogyne incognita, Lecanicillium muscarium, Arthrobotrys oligospora, Dactylella oviparasitica, nematicides

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.1.35.43

* Corresponding author: hussain@af.czu.cz

0030-9923/2017/0001-0035 $ 9.00/0

Copyright 2017 Zoological Society of Pakistan

Introduction

Nematophagous fungi have been extensively studied to control soil-borne plant parasitic nematodes since use of nematicides, soil sterilants and fumigants brings about several ecological and toxicology threats (Jatala, 1986; Santos et al., 1992). Biological suppressive soil has mitigating influence on soil borne pathogens and provoked plant vigor and yield despite of virulence of pathogen, conducive environment and susceptible hosts (Cook and Baker, 1983; Hornby, 1990; Becker et al., 2013; Renčo, 2013). Nematophagous fungi have developed exclusive abilities to parasitize on nematodes in a various way. They are constituted to different taxonomic groups with complex types of prey devices (adhesive knobs or nets, constricted and non-constricted rings) (Barron, 1977; Bird and Herd, 1995; Zouhar et al., 2013). Organisms constitute complex type of networks below soil line with several complex interspecific relationships. However, antagonistic relationship of nematodes and fungi is of more concerned these days (Janson and Lopez-Llorca, 2004). The fungi may harm plant parasitic nematodes in different ways by producing different types of complex trapping devices and specialized organs (Gray, 1987; Jafee, 2004; Zouhar et al., 2013). Plant parasitic nematodes pose a serious danger to several crops worldwide but endoparasitic nematodes (Meloidogyne spp., Heterodera spp., or Globodera spp.) are the most alarming pathogens which reside inside the roots for maximum of their life span (Hussey and Grundler, 1998; Renčo et al., 2012). Due to their feeding behavior and life cycle, it is challenging to control them with nematicides and microbial antagonists while they get established into host tissues (Stirling, 1991; Renčo et al., 201 1). Plan parasitic nematodes despoil all economically important crops though vegetables are more prone to them, (Hussain et al., 2016); causing estimated overall losses of 5%- 43% (Sasser and Freckman, 1987). Overall plant parasitic nematodes cause damages to agriculture and forests amounting to 78 billion US dollars per year worldwide (Barker, 1998; Zhang et al., 2008). Meloidogyne hapla has been a conspicuous vegetable pest in the Czech Republic over the past few years (Nováková and Zouhar, 2009). Losses occur due to prevalence of M. hapla may reach 50-90% of the total crop (Nováková and Zouhar, 2009). More specifically, production losses have been reported for carrots as well as parsley grown in the sandy soils of the Elbe lowland in the Czech Republic (Douda et al., 2010).

Although scientists are struggling to control soil borne pathogens especially plant parasitic nematodes by introducing different biocontrol agents such as fungi, bacteria and predatory insects but main problem is insufficient knowledge on ecology. Biocontrol agents could be more advantageous including germination, proliferation, sensitivity to population variation, and control efficiency if they apply in the same ecosystem where isolated.

The objectives of our study were: 1) to evaluate the potential of the fungi against conspicuous pathogen, M. hapla in laboratory and greenhouse conditions; 2) to determine whether the parasitism was restricted only to eggs or juveniles of nematodes.

Materials and Methods

Nematode culture

Nematode galled roots of tomato plants from our greenhouse were collected and eggs masses were isolated and a single egg mass was used to establish a nematode population. Eggs were extracted from 45-days old galled tomato seedlings roots by using 0.05% NaOCl. Extracted eggs were gently washed with tap water to remove NaOCl (Hussey and Barker, 1973). Meloidgyne hapla specie was identified based on morphological and morphometrical characteristics (Eisenback, 1985). Extracted eggs were placed at room temperature of 25˚C to hatch J2. Eggs and J2 were counted and exposed to test fungi.

Fungus culture

Lecanicillium muscarium was isolated from egg masses of root knot nematodes collected from twenty vegetable fields of tomato, eggplants, and carrot whereas other fungi (Arthrobotrys oligospora, Dactylella oviparasitica, Clonostachys rosea) were isolated directly form the soil collected from localities of Semice (N 50.16265 E 14.89643) and Litol (N 50.18404 E 14.83742) in Central Bohemia, Czech Republic by soil dilution method. Stropharia rugusoannulata isolate was obtained from commercial production of mushroom seed company, the Mycelium Wolf. Uprooted roots with combined infection of nematode and fungi were observed. The black colored egg masses were associated with fungi. On uprooting the plants, roots with shared infection of nematode and fungi were observed. The egg masses were associated with fungi with black color. These egg masses were inoculated axenically on petri plates containing potato dextrose agar (PDA) amended with streptomycin at 1g/L after surface sterilization with 0.5% NaOCl for 2 min (Singh and Mathur, 2014). Petri plates were incubated at 25°C±2°C for ten days. Fungal colonies were isolated and purified by repetitive sub culturing, and later identified and confirmed by using molecular technique polymerase chain reaction (PCR) in laboratory.

All tested fungi were grown on potato dextrose broth (PDB) and kept under room temperature on orbital shaker. After two weeks, fungal mycelia were harvested and culture filtrates were prepared in distilled water and standardized. Each isolate was replicated five times and experiment was repeated once to authenticate our findings.

In-vitro studies

Effect of fungi on egg parasitism

Each fungus isolate was inoculated to the center of Petri dish containing PDA medium amended with antibiotic streptomycin at 1g/L. After inoculation, plates were incubated at 25˚C for one week, and each plate was spread uniformly with 100 M. hapla eggs. Five replicates for each fungus were used whereas eggs without fungi were kept as control for comparison. Evaluation of fungus was noticed after 24, 48, and 72 h and percent egg parasitism was measured by staining with cotton blue and counted under Stereo-binocular microscope. Eggs either infected by direct penetration of hyphae or disintegration of their contents were considered as infected (Khan et al., 2006; Singh and Mathur, 2010). Eggs with live J2 and hatched J2 from eggs were counted as viable.

Effect of fungi on nematode activity and mortality

Five mL of each fungal culture filtrate and 1 mL of nematode suspension containing 100 J2 were placed in sterile Petri plates. Nematode activity was noticed after 24, 48, and 72 h. Fungus efficacy was noticed according to percentage of paralyzed nematodes. Rigid, elongated, and bent nematodes were considered as immobilized if they do not react after probed with fine needle (Cayrol and Pijarowski, 1989; Singh and Mathur, 2010). Revival test was run by centrifugation for 3 min at 1000 rpm, and incubated in sterile water for one day. If nematodes were found inactive after one day, they were considered as dead. Five replications were used in this study.

Greenhouse studies

This experiment was carried out in greenhouse of Czech University of Life Sciences, Prague, Czech Republic. The experiment contained two parts i.e.; one was designed for M. hapla eggs while the other was constructed for second stage juveniles (J2). To investigate the effectiveness of fungi against nematodes, susceptible variety of carrot “Darina” was used. Based on initial inoculum, the experiment was categorized into two. In one experiment freshly extracted nematode eggs (500 eggs/pot); while in the other, one day old second stage juveniles (500 J2/pot) were used. For both experiments, one carrot seedling aged of two weeks was used and one week later inoculated simultaneously with fungi and nematodes eggs and J2 respectively.

Mycelia from all of five fungi were harvested from PDB media, weighed and standard solution (w/v) was prepared in distilled water. 20 ml of the 30% (w/v) solution of each fungus was pipetted on top of soil in each pot. In the control, nematodes were applied without nematophagous fungi. The pots were placed in completely randomized design (CRD) with five replications on a bench in a greenhouse. The pots were irrigated after two-day intervals throughout the period of study. The daily temperature ranged between 25° and 28°C.

Data collection

After 75 days, plants were carefully removed from the pots and their roots were cut off from the shoots. The roots were gently washed under tap water and blotted dry. Fresh root shoot weights and lengths were recorded. The numbers of galls were counted under Stereomicroscope at magnification 40X. For determination of total nematode population, the eggs were obtained from the roots by using 0.5% NaOCl solution passing through a sieve with pore size of 74 and 25mm (Hussey and Barker, 1973; Hussain et al., 2016). The juveniles were extracted from the soil of each individual plant from their respective pots (Whitehead and Hemming, 1965). The total number of eggs and nematodes in soil were also noted and constituted the total nematode population. The potential of nematophagous fungi was evaluated in terms of nematode reproduction factor (Rf); where Rf is the final nematode population at harvest (Pf) divided by the initial nematode population (Pi) at inoculation. The reproduction was calculated by dividing the final population by 500 in both experiments.

Statistical analysis

All experiments were repeated once. All pool data from two experiments were subjected to analysis of variance (ANOVA). The means were compared by the Duncan Multiple Range Test (DMRT) at P=0.05 by using software Statistics 8.1.

Results

In vitro study

Lecanicillium muscarium had more parasitic activity in terms of M. hapla egg infection after 72h followed by S. rugosoannulata (89%). Whereas other species also showed better results in egg infection. There were a few eggs able to hatch while exposed to fungi L. muscarium and D. oviparasitica (Table I). It was seen that L. muscarium and D. oviparasitica invaded eggs in early stages of their development and parasitized them with emerging juveniles. In case of S. rugosoannulata J2 were trapped by acanthocytes which can be seen in Fig. 1. During early stage of infection, fungal hyphae completely occupied the embryo within egg and consumed all contents of eggs leaving them empty. None of fungi produce their resting spores such as chlamydospores etc in nematode eggs, although conidia were seen occasionally in Petri plates. Eggs containing second stage juveniles were escaped first but later they were preyed by fungi as well. In case of inactivity and mortality of nematode juveniles, all fungi behaved excellent but the percentage of egg and J2 parasitism was higher in case of L. muscarium and S. rugosoannulata. After revival test, a few juveniles were able to stay alive which is more obvious in Table I.

Greenhouse study

In greenhouse experiments, all tested fungi significantly suppressed M. hapla reproduction by reducing number of galls and egg masses on roots and J2 in soil in comparison to control (P < 0.05). The highest reduction of final nematode population in soil treated with L. muscarium have been found followed by S. rugosoannulata and D. oviparasitica (Table II). Of the media tested, L. muscarium gave the best results of biomass production for most fungal species. It is also found that L. muscarium colonized the gelatinous matrix and preyed upon eggs more than J2 of nematodes. The behavior of other fungi in both studies of M. hapla egg and J2 infection was also similar but less pronounced to L. muscarium (Table II). In comparison to others, A. oligospora was found less reactive whereas C. rosea and S. rugosoannulata showed better results in parasitism of eggs and J2 as well (Table II). In general, plant development was more in case of fungal treatment as compared to their respective controls. The growth of shoots and roots were improved in all of treated plants (Fig. 2). In comparison, L. muscarium proved to be the best

Table I.- Parasitic efficiencies of fungi on eggs and juveniles (J2) of root knot nematodes, Meloidogyne hapla

Fungi/ Treatments | Egg infection (%) | Inactive nematode at various interval (%) | Active neatodes (%)*
| 24h | 48h | 72h | 24h | 48h | 72h
A. oligospora | 24.6± 3.36c | 54.4± 5.37c | 88.6± 2.07b | 53.8± 7.40 a | 72.2± 5.81 b | 84.8± 3.70 c | 6.8± 1.48 b
D. oviparasitica | 28.4± 3.51bc | 63± 2.74ab | 88.2± 1.30b | 55± 3.81 a | 74.6± 2.70 b | 85.8± 3.63 bc | 7.8± 0.84 ab
S. rugosoannulata | 32.6± 3.51b | 58.4± 5.13bc | 89.4± 2.88ab | 53.6± 5.50 a | 72.6± 3.05 b | 89.4± 2.70 b | 7± 1.58 b
C. rosea | 30.8± 4.82b | 64.6± 1.52a | 86.4± 10.29b | 52.4± 4.67 a | 70.2± 4.21 b | 86± 2.92 bc | 9.6± 2.41 a
L. muscarium | 42± 2.24a | 66.2± 2.59a | 95.6± 2.41a | 55± 3.39 a | 83.2± 3.56 a | 95.8± 2.39 a | 2.4± 1.67 c
Control (PDB) | 00 | 00 | 00 | 00 | 00 | 00 | 00
Control (Water) | 00 | 00 | 00 | 00 | 00 | 00 | 00

* After revival in water; Data are mean of ten replications. Means in each column with different letters differ significantly according to Duncan’s Multiple Range Test at P = 0.05.

Table II.- Influence of nematophagous fungi to Meloidogyne hapla reproduction and plant growth parameters of carrot in greenhouse, 75-days after inoculation with an initial population density (Pi) of 500 eggs and 500 J2 per plant

Fungi/ Treat-ments | Galls/ root syst- em* | Egg masses/ root syst- em* | Egg produ ction/ root system | J2 prod- uction/ 100cc of soil | Repro- duction factor ** (Pf/Pi) | Fresh shoot wt. (g) | Fresh root wt. (g) | Fresh root length (cm) | Fresh shoot length (cm)
After inoculation of Pi of 500 eggs/plant
A. oligo-spora | 11.0± 0.11b | 5.00± 0.39b | 138.± 1.3b | 215.0± 0.45b | 2.42± 0.023b | 13.0± 0.98c | 16.8± 1.2c | 9.14± 1.2b | 36.2± 3.5d
D.ovip- arasitica | 9.00± 0.14b | 7.00± 0.48b | 122.± 1.2b | 212.0± 0.42b | 2.36± 0.33b | 14.0± 0.92bc | 28.8± 2.3b | 9.29± 1.23b | 37.2± 3.7cd
S. rugos-oannulata | 8.00± 0.32b | 5.00± 0.36b | 111.± 1.09b | 175.0± 0.31b | 1.97± 0.01b | 15.0± 0.87b | 14.8± 1.4cd | 10.0± 0.9ab | 39.4± 4.1c
C. rosea | 7.00± 0.22b | 4.00± 0.01 b | 118.± 1.02 b | 199.0± 0.38b | 2.23± 0.03b | 13.1± 1.0c | 13.1± 0.98d | 10.0± 1.3ab | 44.1± 4.3b
L. musc- arium | 3.00± 0.03 c | 2.00± 0.02c | 99± 0.99b | 102.0± 0.36b | 1.22± 0.04b | 17.8± 2.1a | 56.0± 3.4a | 11.0± 0.97a | 50.0± 5.2a
Control | 40.0± 1.9a | 27.00± 1.1a | 2671± 2.1a | 1518± 0.35a | 20.52± 1.2a | 10.4± 1.2d | 8.5± 1.2e | 4.43± 0.77c | 26.1± 2.1e
After the inoculation of Pi of 500 J2 /plant*
A. oligo- spora | 9.00± 0.23b | 5.00± 0.34bc | 241± 2.1b | 818.0± 1.34b | 8.66± 0.98 b | 12.7± 1.2b | 17.2± 1.2c | 8.10± 0.87b | 35.2± 1.23c
D. ovipa- rasitica | 10.0± 0.34b | 7.00± 0.31b | 222± 2.3b | 713.0± 1.44b | 7.57± 0.99b | 13.2± 0.99b | 29.7± 2.1b | 8.00± 0.68b | 35.2± 1.34c
S. rugoso- annulata | 8.00± 0.07b | 6.00± 0.41c | 211± 1.99b | 612.0± 1.41b | 6.54± 0.93b | 13.0± 0.98b | 16.1± 1.4c | 8.3± 0.67b | 37.0± 1.20c
C. rosea | 9.00± 0.03b | 5.00± 0.23 bc | 208± 2.3b | 519.0± 1.43b | 5.60± 0.77b | 13.1± 0.93b | 13.2± 1.25d | 9.50± 0.3ab | 43.4± 1.24b
L. mus- carium | 7.00± 0.02b | 5.00± 0.13c | 163± 1.98b | 319.0± 1.23b | 3.51± 0.67c | 17.2± 0.88 a | 55.2± 1.68a | 9.80± 0.37a | 49.2± 1.51a
Control | 38.0± 1.42a | 28.00± 1.2a | 3953± 2.3a | 1099± 2.4a | 18.89± 0.99a | 11.2± 1.2c | 9.20± .89 e | 5.10± 0.32c | 25.2± 1.6 e

Data are mean of ten replications; Means within a column followed by the same letter are not significantly different according to Duncan’s Multiple Range Test at P = 0.05.

*Gall and egg mass indices: 0-5 scale; where 0 = no galls or egg masses, 1 = 1-2 galls or egg masses; 2 = 3-10 galls or egg masses; 3 = 11-30 galls or egg masses; 4 = 31-100 galls or egg masses, and 5 = > 100 galls or egg masses per root system (Quesenberry et al., 1989).

**RF, Reproduction factor whereas Pf is final nematode population density divided by initial nematode population density.

nematophagous fungi against M. hapla followed by C. rosea and S. rugosoannulata. The greater root weight and longer shoot length in case of carrot resulted better developed roots systems rather than an increased weight of galls on roots. In treatments receiving nematodes, roots were distorted and bushy with galls. The treated roots were in proper shaped with higher contents of food as compared to control (Figs. 2 and 3). In case of L. muscarium root shoot weights and lengths were improved in both forms of inoculums; eggs and J2, which is more prominent in Table II. Furthermore, the higher percentage of root shoot lengths and weights with low reproduction factor declared that L. muscarium was more potent than other fungi in experiments (Table II). A very few nematodes were recovered from the roots of the plants and their respective pots, treated with fungal species. Roots were observed healthy and sturdy with fabulous shape, and were eatable.

Discussion

All of fungal species produced excellent results in parasitism of eggs and juveniles. In our study, five fungi including A. oligospora, D. oviparasitica, C. rosea, S. rugosoannulata, and L. muscarium were isolated from vegetable fields in association with egg mass of M. hapla, where A. oligospora Fresen, is a nematode destroying predacious fungi (Shepherd, 1961) which develop a characteristic organs of three dimensional networks sticky hyphal anastomosing loops to capture prey (Nansen et al., 1986). Moreover, the fungus has also been reported to control animal parasitic nematodes (Descazeaux, 1939; Roubaud and Deschiens, 1941; Soprunov, 1958; Pandey, 1973; Gronvold et al., 1984; Gruner et al., 1985).

Dactylella oviparasitica (Stirling and Mankau, 1978) was first isolated from Meloidogyne egg masses collected from peach orchards in the San Joaquin Valley, California. This fungus proved to be more aggressive against Meloidogyne eggs rather than J2 and any other nematodes spp. D. oviparasitica is believed to produce appressoria as well as chitinase enzyme (Stirling and Mankau, 1979) which might be responsible for successful penetration into eggs, eggmasses and J2. Clonostachys rosea is a facultative saprophyte (Schroers et al., 1999) and potential bioagent which is extensively being used against plant pathogenic fungi i.e., Botrytis cinerea (Morandi et al., 2003), and plant parasitic nematodes (Zhang et al., 2008). Fungus is capable to produce large number of conidia in a very short time which stick to nematodes and proliferate into body cavity after germination. The fungus is believed to produce hydrolytic enzymes such as proteases, collagenase, and chitinase which may be involved in nematode cuticle penetration and host cell degradation (Tunlid et al., 1994; Åhman et al., 2002; Yang et al., 2007). S. rugosoannulata is related to nematophagous fungi found in woods, grasslands and composts (Luo et al., 2006) which produce specialized structure of large spike cell called acanthocytes (Farr, 1980), which mechanically destroy nematodes eggs as well as J2. Lastly, L. muscarium is an entomopathogenic fungi which produce mucilaginous matrix enables to attach with host surface. Fungal conidia produce fine hyphae on germination of conidia and hydrolytic enzymes including chitinases and gluconases which help fungi to penetrate nematode eggs and J2 mechanically or by enzymatic actions (Yang et al., 2005). Observation on the proliferation of these fungi in M. hapla eggs and J2 showed that appressoria were formed by most of fungi especially in case of D. oviparasitica. Appressoria is an important device of fungi to get attachment and penetration in host (Emmett and Parberry, 1975). In our understanding, plant parasitic fungi most commonly penetrate their hosts mechanically but enzymatic penetration or combination of both could be happened in some fungi (Emmett and Parberry, 1975). Most of entomopathogenic fungi such as L. muscarium produce hydrolytic enzymes which enable them to penetrate cuticle of nematode eggs and J2 (Gabriel, 1968; Latge, 1974; Leopold and Samsinokova, 1970; Shinya et al., 2008). Mechanical penetration was also observed in most of fungi tested, but since chitin is the main constituent of Meloidogyne egg shells (Bird, 1976), production of chitinase in culture indicates that enzymatic penetration was also occurred. Behaviour of A. oligospora was irregular because it had better results in mortality of J2 after 24h and 48 h but after 72h it was not quite significant which might be its growth factor in culture. Growth of A. oligospora was slowest in culture media due to which it did not have prominent parasitic effects on nematode eggs and J2. But it is believed that A. oligospora produce three dimensional hyphae trap as well as toxins which paralyze nematodes very effectively (Bird and Herd, 1995; Nansen et al., 1986). Mat of mycelium was observed around eggs and J2 in case of C. rosea and S. rugosoannulata which was clear evidence of their successful penetration and parasitism. Both are referred to nematophagous fungi producing mechanical traps (Barron, 1977; Zouhar et al., 2013) as well as hydrolytic enzyme which enable them to dissolve cuticle and produce successful infection (Zhang et al., 2008). These fungi were assessed against M. hapla in greenhouse. All fungi had enormous effects on plant growth as well as nematode reproduction including gall and egg masses indices and egg and J2 production in roots and soil. L. muscarium proved to be more effective in reducing the infection level of nematodes as compared to control plants. Agility of this fungus in parasitism was more in nematode eggs rather than J2 which could be its multiple way of action. It is believed that L. muscarium produce very fine hyphae to rupture the cuticle of nematode eggs mechanically; also produce chitinases which facilitate in penetration of egg shells and J2. Furthermore, this fungus was also documented to stimulate induced resistance in plants (Hirano et al., 2008). Presence of number of galls on roots and J2 in soil of control plants validated the studies done by Larkin et al. (1993), which stated that maximum population of nematodes was found in fumigated soil rather than non-fumigated. Efficiency of L. muscarium may also be correlated to temperature which produces high level of conidia and enzymes, in wide range of temperature (5-30°C) with an optimum at 25C (Fenice et al., 1996; 1997). Below the soil line, several biological interactions are happening which are necessary to maintain ecosystem. These nematophagous fungi produce different types of mechanical traps for nematodes which may restrict the free movement of nematodes in soil (Jensen et al., 2004). In this way, nematodes could have limited chance to approach root zones to get penetrated into host plants. Nematophagous fungi also produce different types of enzymes for example chitinases, proteases, amylases etc to pin down nematode population. Overall all fungi behaved excellent in our greenhouse studies but L. muscarium, S. rugosoannulata and C. rosea produced best results in reducing nematode reproduction factor (Pf/Pi) and escalation of plant growth. Root systems of treated plants were colonized with fungi when observed under microscope which was clear picture of protecting shield against plant parasitic nematode (Koike et al., 2001).

ConclusionS

All of fungi were highly effective at managing root knot nematode population up to maximum extent. Among fungi, L. muscarium had greater potential to reduce infestation level in soil up to greater extent which happened due to their multiple parasitic actions; production of enzymes, fine hyphae and induced resistance at wide range of temperature. It was further documented that the efficient antagonistic relationship between fungi and nematodes had positive effects on plant growth compared to controls.

Acknowledgement

We are thankful to Czech University of Life Sciences Prague for providing funds to conduct part of this research under project number PROJ201500056.

Conflict of interest statement

The authors have declared no conflict of interest

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