Hirsutella thompsonii and Pochonia chlamydosporia ( Syn . Verticillium chlamydosporium ) Mycelia Growth and Predation on Panagrellus redivivus

This research aimed to evaluate the nematophagous ability of 4077-Verticillium chlamydosporium var. chlamydosporium and 4466-Hirsutella thompsonii isolates and relate mycelia growth to the influence provoked by movement of nematodes. Each fungus grew in PDA (potato, dextrose, agar) medium end up to pure colonization. Then, ten mycelia plugs of 8 mm diameter were removed from colony borders and transferred to the center of ten Petri plates containing water-agar 2% medium. These plates were previously divided into four quadrants that received a number of 25 individuals of free-living nematodes (Panagrellus redivivus), composing a total of 100 nematodes per plate. Evaluations started after 24 hours of interaction, considering predation percentage and mycelia growth as stimuli of nematodes presence. Results showed growing predation performance to both isolates, being higher for V. chlamydosporium var. chlamydosporium since from first evaluation time, controlling more than 50% of nematode population initially added. Its predation potential was 39.2%, 38.4% and 48.35% higher than H. thompsonii at first, second and third evaluation day, respectively. Generally, nematodes did not stimulate mycelia growth, unless for H. thompsonii at 72 hours of interaction compared to control plates (without nematodes). Stress resulting from isolates transference from PDA to water-agar 2% resulted in sparse mycelia growth and it could have affected the predation performance of H. thompsonii that controlled nematodes in low levels throughout experiment. Independently of predation level, pictures revealed that both isolates has ability to control P. redivivus through hyphae penetration.


Introduction
Biological control of parasitic nematodes represents a viable strategy to minimize chemical application that causes lots of damage in soil biotic microorganisms (Morandi et al., 2009).Among nematophagous microorganisms, fungi are preferred due to their ability on capture and digest nematodes, even free-living ones.In this process, hyphae modifications may occur in some species in order to hold nematodes before preying, changing growth pattern (Chen & Dickson, 2004).Many fungal species have been continually investigated as a control strategy alternative (Morandi et al., 2009) and the facility in manipulating Panagrellus redivivus turned its use in laboratory trials very common (Sautter et al., 2007).
Members of the genera Hirsutella and Verticillium are filamentous fungi that compose a part of a very diversified group that should succeed better in soil once they can grow even in regions where nutritional sources are scarce (Morley et al., 1996).Some species belonging to Hirsutella showed ability as biological agents under greenhouse trials (Xiang et al., 2007), as well as Pochonia chlamydosporia (syn.Verticillium chlamydosporium) that releases nematicidal substances (Khambay et al., 2000).Both genera are saprophytic and P. chlamydosporia plays an important role as plant growth promoter (Macia-Vicente et al., 2009).
As a diversified group, fungi have different feeding pattern, saprophytic absorbs nutrients directly from organic matter and mycorrhizal by association with host plants (Rousk & Baath, 2011).Fungi survival depends on external carbon source, such as organic acids released by root exudates (Broeckling et al., 2008).Likewise, nematodes can stimulate the activity of some saprophytic fungal species being a nitrogen source to their development (Barron, 2003).Nematodes also may induce modification in mycelia patter growth, as trap formation (Gronvold et al., 1996).
The present study aimed to determine predation of the free-living nematode Panagrellus redivivus by 4077-Verticillium chlamydosporium var.chlamydosporium and 4466-Hirsutella thompsonii isolates and relate mycelia growth to the influence provoked by movement of nematodes.

Material and Methods
Individuals of Panagrellus redivivus were maintained in a pasty mixture of oat flour and distilled water.Those nematodes climbing the pots were removed with a spatula and put into a Bequer containing distilled water, then they were shaken to get cleaned from flour debris.Afterwards, the solution was poured under a 400 mesh sieve.During this process, nematodes got separated from solution, thus facilitating their capture via pippets.Nematode population was established to allow the extraction of approximately 25 specimens in 20 µL of distilled water using an electronic micropipette.
To stimulate growth of mycelia and predation tests, agar plugs (8 mm diameter) of pure mycelia grown in PDA were transferred to the center of 10 Petri dishes containing water-agar (2%) medium.All Petri dishes were divided into four quadrants and, when mycelia radius expanded 1.5 cm, each quadrant received a media of 25 nematodes.Control dishes were constituted only by pure mycelia without added nematodes.Plates were stored into body oxygen demand (BOD), at 23.3 ºC, under dark condition, during experiment.
After 24 h of nematode-fungi interaction, mycelia growth measuring started in treatments and controls, during three days.This evaluation considered four radial growing (two diametrically opposite) measured with graduated ruler to establish mycelia area colonizing the plates.
For treatments, nematode predation percentage was also analyzed by counting the number of dead nematodes from the population initially added to the plates.Predation status was considered from first colonization signal, as single hyphae penetration, from what on, flight was not possible and full control depended on the time.
Pictures were taken using a cell phone camera of 12 Mp coupled to an optical microscope (Nikon, model ECLIPSE E100 LED).Data were analyzed by a variance analysis (ANOVA) with a significance of P < 0.05 Tukey media test was applied at 5% of probability using SISVAR 5.6 ® statistical program (Ferreira, 2011).

Results
Many studies suggest that nematophagous fungi grow more under high nematode population levels in soils what would lead to a successful rhizosphere colonization.Nevertheless, V. chlamydosporium var.chlamydosporium showed no significant difference in its mycelial growth under nematode presence throughout the experiment (Table 1).H. thompsonii grew more in treatment than in its control with significant difference only after 72 h of incubation.Bourne and Kerry (1999) and Mauchline et al. (2004) who concluded that there is no simple relation between mycelia extent and predation levels.
Mycelia growth and egg parasitism of M. javanica by 18 isolates of P. lilacinus and three isolates of P. chlamydosporia under five temperatures influence revealed statistical difference (Stroze et al., 2013).However there was no apparent relation between mycelia growing and egg parasitism level at any incubation temperature for P. lilacinus.Isolates of P. chlamydosporia did not differ to growing parameter at any temperature and egg parasitism was lower for only one isolate at 20 ºC.
Greenhouse trials had shown an indirect effect of host plants on fungal development on rhizosphere (Bourne & Kerry, 1999).Thus, not only artificial medium plays important interference on fungal performance, but also the root plants to which they need to establish any relation to succeed as control agents.Mauchline et al. (2004) analyzed root colonization of three isolates of P. chlamydosporia in potato and tomato plants, in both, health or infested conditions.Infested treatments consisted in individual presence of nematodes Globodera rostochiensis or M. javanica.Results revealed that one isolate growth was not effected by either nematode species presence.However, one isolate grew more in treatments with M. javanica in both plants.In this study, the presence of P. redivivus did not stimulate V. chlamydosporium var.chlamydosporium growth.These responses suggest high dependence on the fungal isolate to determine how much mycelia growth may be stimulated by different nematode species.
Many studies relate the nematode controlling agents colonization to plant exudate influence or organic matter content preferably than to nematode stimuli.De Leij and Kerry (1991) observed that V. chlamydosporium colonization in rhizosphere depends on fungal isolate and plant species, possibly because different plant species produce and release distinct exudates compounds that can improve or inhibit microorganisms association with roots (Haichar et al., 2014).This lack of stimuli to mycelia growth of V. chlamydosporium var.chlamydosporium matches to findings of Quinn (1987) who established that predation activity is more related to the pressure caused on saprophytic ability by others microorganisms in soil than for nematode population density in soil.Here, fungal isolates were set individually to prey nematodes not being possible to observe predation potential under saprophytic pressure.
Nematophagous fungi are classified as predacious, parasitic and opportunistic.Genus Verticillium contains opportunistic individuals, as Paecilomyces.This group of nematophagous agents are considered better over predacious and parasitic ones, due to their ability to colonize eggs and cysts released in soil, preventing larger juvenile infection (Siddiqui & Mahmood, 1996).In this study, V. chlamydosporium var.chlamydosporium preyed on P. redivivus, a free-living nematode whose life cycle does not have eggs or cysts.Therefore, this fungal specie display a role as predacious agent too, once it captures nematodes by hyphae, a characteristic from predacious species.
A chemical study performed by Niu et al. (2010) detected toxicity of two aurovetin compounds obtained from fermented mycelia extract of P. chlamydosporia on worms of P. redivivus, leading to body disintegration.This chemical support may explain the better performance of P. chlamydosporia on preying P. redivivus compared to H. thompsonii ability (Table 2).
The ability of fungal agents on controlling nematodes depend on several factors like environment conditions, isolate genetic, host plant, nematode specie and go on.Other details are even more specific, since V. chlamydosporium produces proteases that hydrolyses outer layer proteins that compose eggshell of M. incognita (Segers et al., 1994).For such reasons, many studies propose a mixture of distinct fungi species or isolates to enhance biological control activity, considering particularities of each living involved.Isolates mix of P. chlamydosporia assured additive effect on nematodes eggs reduction Mauchline et al. (2004).

Conclusions
Panagrellus redivivus did not stimuli mycelia growth of V. chlamydosporium var.chlamydosporium at any time while H. thompsonii grew significantly more in presence of nematodes only at third evaluation day.
Both isolates were able to prey nematodes.Predation levels of V. chlamydosporium var.chlamydosporium were superior to H. thompsonii in all evaluation times.