Daily Indexes for Predation and Growth of Nematophagous Mushrooms Species of Hohenbuehelia (Pleurotaceae) on Panagrellus redividus

Biological control is a method of controlling pests through the use of other living organisms. The purposes of this study were to test Hohenbuehelia species as biological control agents against Panagrellus redivivus in vitro, evaluating nematodes influence on mycelia growth; establishing daily indexes for predation and growth and setting predation percentage. Five species previously identified as 436-Hohenbuehelia mastrucata (Nematoctonus hamatus), 528-H. bullulifera (not described so far), 581-H. paraguayensis (N. sp.), 582-H. sp. (N. sp.) and 631-H. portegna (N. campylosporus) were submitted to anamorphic purification directly from basidioma. Afterwards, 100 nematodes were added to each pure colony for predation test. Evaluation started right after 24 hours of nematode-fungus interaction. Immobilized and/or penetrated nematodes were counted and mycelia growth was measured. Results were subjected to variance analyses. Hohenbuehelia mastrucata had the best performance in growth speed, followed by H. portegna and H. paraguayensis; Nematodes multiplyied much but none specie grew more as an influence of their movement under mycelium, however all species formed trap devices and some of them produced adhesive or repelent substances. Trap devices were formed in control plates also. The plates of H. paraguayensis without nematodes grew more than treatments. Cumulative predation of H. portegna was the highest at 24 (195.5%) and 48 hours (235%). At the last evaluation day, H. paraguayensis preyed the same amount (185.75%) than H. portegna, followed by H. mastrucata (109.51%). Resulst of predation daily indexes displayed chronological activity for each isolate, where H. portegna was very reactive at first 24 hours, H. mastrucata raised its predacious activity in 48 hours being constant from this time on and H. paraguayensis pointed out itself at 72 hours. Other species presented low predation and growth indexes throughout experiment.


Introduction
Phytonematodes attack causes reduction in productivity what can reach alarming levels.They are microscopic worms present in almost all soils, having several plants as hosts, including weeds.Their dissemination occurs by soil and roots transport.Chemical products are usually applied to control them, but a growing concern about human and animal health and environmental care have reduced their use (Ferraz & Brown, 2016).Nematode control is a very difficult task resulting in increasing interest on alternative methods, such as biological control.
Biological control is a natural process occurring in all habitats, nevertheless it can be introduced in agricultural areas by using a living organism (Tranier et al., 2014) to keep low a population level of another organism (Agbenin, 2011).Amongst several potential agents, fungi have been traditionally studied (Vega et al., 2009).
Hohenbuehelia genus contains nematophagous species belonging to Basidiomycota phylum, presenting clamp connections as intrinsic characteristic (Babu et al., 2014).They are filamentous endoparasites that adhere nematodes to a trapping apparatus to immobilize them and penetrate their cuticle, invading the body and digesting its nutrients (Tranier et al., 2014).Barron (2003) observed ligninases and cellulases production, suggesting that Hohenbuehelia (syn.Nematoctonus) species decompose woods naturally and nematodes predation is a subset to get nitrogen supplement.It happens due to the high carbon to nitrogen ratio in woods composition maintaining its stability that reduce degradation by fungi (Piskur et al., 2009).Therefore, this genus performs two important functions to ecosystem: wood decomposition and pests control (Mankau, 1980).Further, saprophytic ability turns biological agents into a promising control strategy (Nunes et al., 2010) and nematode trapping fungi present no specificity to host so any nematode in soil can be preyed by them (Askary, 2015).Koziak et al. (2007) confirm that Nematoctonus genus are found in agricultural soils and contains predators and parasitoids species, unlike other nematophagous genus that generally contemplates only one control profile.
Panagrellus redivivus is a free-living nematode visible at naked eye during adult stage that reproduces very fast (Sautter et al., 2007) and for that reason it is frequently used in laboratory trials.
The present study aimed at analyzing the potential of Hohenbuehelia species as biological control agents in vitro against Panagrellus redivivus, identifying a relation between nematode predation and mycelia growth, establishing daily indexes for predation and growth and setting predation percentage.We hypothesize that the more isolates grow the more nematodes are preyed by them.
Basidiocarp collected were in a very dirty condition and with low spore formation so they were purified directly from gills and gelatinous context of fresh or dried basidiocarp, right after superficial disinfestation method.The pieces were chosen and cut with needle from areas apparently free from contamination.
First, small pieces of gills were taken using a needle and driven to sterilized laminar flow where got submerged in sodium hypochlorite (1%) during three minutes for disinfestation.In sequence, they were put into a recipient with alcohol (70%) during 30 seconds to remove hypochlorite excess, followed by three washings in sterile water.
After superficial purification, basidiocap fragments were carefully placed on Petri plates with PDA (potato, dextrose, agar), medium, sealed with Parafilm and stored into BOD (body oxygen demand) at 25±2 o C. Purified mycelia colonies were detected visualizing clamp connections on septum of hyphae.

Predation Test and Mycelia Growth Measuring
For predation test, eight agar discs (8 mm diameter) were removed from borderer mycelia of each purified colony grown in PDA medium and replaced to the center of eight 9-cm-diameter Petri plates containing water-agar (2%) medium.
A pasty mixture of oat flour and distilled water was done to maintain nematodes.Those were collected with a spatula when climbing the boundary of pots, mixed in water and poured under a 400 mesh sieve for cleaning and separation from flour.
Four plates represented the control (without nematodes) and four the treatments (fungus + nematodes).All plates were divided into four quadrants marked on background with marker pen to allow an accurate counting of dead nematodes.In treatments, each quadrant received individually 20 µL of distilled water containing a media of 25 Panagrellus redivivus.In sequence, plates were stored into BOD at 25±2 o C throughout evaluation period.
Four measures of radial mycelia growth per plate were done aided by a ruler.
Analysis on predation percentage and mycelia growth started passed 24 hours of nematode-fungus interaction up to third day.Nematode were considered preyed from single hyphae penetration or when glued on hyphae.New borne juveniles were counted together.
Pictures of preyed nematodes and of trap formation were taken using a cell phone camera of 12 Mp coupled to an optical microscope (Nikon, model ECLIPSE E100 LED).

Statistical Analysis
Obtained data was transformed by √(x + 1) and subjected to analyses of variance (ANOVA).In the case of significant results (P < 0.05), Tukey test (5% error probability) was employed to compare averages using SISVAR 5.6 ® program (Ferreira, 2011).

Results
The isolate 582-H.sp.could not be identified due to lack of spores, an indispensable structure used for identification.
Most isolates grew in a low rate, so during mycelia purification they were subculture many times to new sterilized PDA medium to conclude the process in order to keep them away from contaminations in early stages.
Each colony took different time to grow on agar before nematodes addition (Table 1).H. mastrucata had the fastest colonization profile (relation between time and growth).Note.* Means followed by the same letter in the same column did not differ significantly from each other by Tukey test at 5% of probability.
Table 2. Growth (cm) comparison between treatments (with nematodes) and controls (without nematodes) of Hohenbuehelia spp.after 24, 48 and 72 evaluation periods Note.* Means followed by the same letter in the column did not differ significantly from each other by Tukey test at 5% of probability.
Hohenbuehelia portegna predation stood out from first 24 hours of interaction (Table 3).This isolate released colorless substances with apparent sticky property that attached nematodes to its hyphae precluding any movement out of mycelia, ascribing a great reactiveness.
jas.ccsenet.According to Ren and Yao (2013), biomass fungi production decreases in poor nutritional substrate.However, plates containing nematodes as a nutritional source presented the same growth than control ones.Each treatment received about 100 specimens that moved freely above hyphae of most species enabling an easy capture.This amount possibly satisfied fungi nutritional requirement readily (Soto-Barrientos et al., 2011), not being necessary further growth, so as to H. paraguayensis that grew more in nematodes absence at all evaluation times (Table 2).
The low growth rate of H. paraguayensis and H. bullulifera (Table 1) could turn difficult their mixture with other nematophagous fungi presenting additional modes of action and chemical compatibility to other nematodes species to improve biocontrol technique, because these species would be probably overgrown by fungal as Trichoderma spp. or be antagonized by them (Szabó et al., 2012).
The presence of P. redivivus by itself did not stimuli fungi growth.Andersson et al. (2014), studied genes expression during infection process and proved chemical specificity between fungal-nematode interactions.However, nematode-trap fungi do not appear to have specificity to nematode host (Kerry, 2000).Our results suggest that trap formation does not imply in higher predation since the specie that formed more traps (H.bullulifera) was one that less preyed nematodes (Table 3).In addition, as fungi developed traps even in plates without nematodes, modifications on mycelia pattern and on growth were definitively not influenced by nematodes movement.
Thorn and Barron (1986) observed that N. brevisporus did not formed adhesive knobs from conidia in the presence of nematodes while N. angustatus could not adhere its spore to nematode body.Their conclusion shows that even both species are predators they were not stimulated by nematodes.Gronvold et al. (1996) observed trap formation of Duddingtonia flagrans being influenced by hyphae aging, with better activity at 30 o C than 20 o C, enduring for two or three weeks, period from what on trap inductility reduced.
The authors also guarantee that larvae migration (Ostertagia ostertagi) was indispensable for trap formation and as their movement reduces at 10 o C it explicates low trap formation at this temperature, though their work did not considered control plates to conclude whether trap would be induced only in plates with nematodes.
Despite fungi did not increase their growth in response to nematodes movement under mycelium, some of them produced metabolites, attracting or repulsing them (Figure 4).Mankau (1980) points out that several nematophagous fungal species release substances from infection pegs (Figure 2A) that may intoxicate nematodes by indirect contact and immobilize them promptly for predation.Drechsler (1946) reveals a common 8-shaped structure (Figure 2A) formed at the hyphae tip as an active glandular cell responsible for producing adhesive substances in Nematoctonus genus with a possible occurrence of a second or third adhesive knob.During microscopic observations, this tiny apparatus was only found twice for H. portegna under a cadaver, one time for H. bullulifera and none time for other species.Thorn & Barron (1986) observed nematodes predation by N. cylindrosporus even no adsehive knobs was reported for this specie, similarly to H. paraguayensis.According to them, H. mastrucata may be predatory as well as endoparasite, which adhesive knobs are few or very tiny.We did not find adhesive knobs in H. mastrucata mycelia.Babu et al. (2014) studied predation pattern of N. robustus (H.grisea) and observed nematodes attached to hyphae by hourglass shaped adhesives knobs, being penetrated later, just as noticed for H. portegna (Figures 2A  and 2B).
These structures hold nematodes before body penetration.A single knob would be attached one juvenile avoiding its movement Drechsler (1946).However, we rarely saw such structures what makes us to believe that each glandular cell may produce large amounts of or very toxic adhesive substance(s), since more than 100 nematodes got sticked on hyphae in 24 hours.Interestingly low or no motility was noticed to nematodes attached by H. portegna.In fact, the presence of adhesive knobs enables a single hyphae system to capture large numbers of nematodes (Thorn & Barron, 1986).
The presence and activity of adhesives knobs classify a parasitic relationship.Misleading classification related to the mode of action, allows a fungal that presents adhesive knobs to be positionated in at least two different categories: predacious and alternative parasite, beyond being an antibiotic producer (Chen & Dickson, 2004;Lopez-Llorca et al., 2008).Thorn and Barron (1986) performed an identification key for Nematoctonus species and related the presence of adhesive knobs to classify predacious species while the absence of this structure determine endoparasitic species.
Nematodes are sttoped by adhesive spores or hyphae of Nematoctonus species (Thorn & Barron, 1986).High sporulation levels are expected in locals where groups of nematodes are preyed Drechsler (1946), but not a single spore were seen to none isolate colony at any evaluation time, even in area with massive predation (Figure 1A).Herewith, predation success does not rely exclusively on spore formation.
Most field trials on biological control of nematodes using trapping-fungi as a manageable tool utilize spores as potential structures to vehicular them into soil (Giuma & Cooke, 1974;Sahebani & Hadavi, 2008;Noweer & Al-Shalaby, 2014) and keep longer their permanency in it.In this study, Hohenbuehelia species did not present spores formation in asexual state (Nematoctonus).
Not only hypha penetration and spores are known to be involved in nematode paralysis, also chemical substances play an important role.Singh and Mathur (2010) evaluated culture filtrates of 14 fungal species and noticed that all of them presented antagonistic activity against Meloidogyne incognita, in different levels, after 24 and 72 hours of interaction.Likewise, predation of H. bullulifera, H. portegna and H. paraguayensis may have been influenced by chemical compounds releasing.It is known that Hohenbuehelia genus contains endoparasites species that produce toxins (Moosavi1 & Askary, 2015).Probably toxin production differs among species and isolates what ends up in varied control ability.
Degenkolb and Vilcinskas ( 2016) proceed a fermentation protocol to identify secondary metabolites biosynthesized by Nematoctonus robustus (H.grisea).Among them, dihydropleurotinic acid, pleurotin and leucopleurotin showed weak activity against fungal and bacteria, but no nematicidal effect on Caenorhabditis elegans.However, tests against others nematode species were not driven by these authors.
In this study, substances were released on agar not only in plates containing P. redivivus, but also in control plates, indicating physiological stress or natural defense (Girotto et al., 2008).In this last case, these substances probably have properties against others organisms too, increasing the potential of Hohenbuehelia species as biological control agents, since many species from Basidiomycota are expected to produce antibiotic molecules (Robins et al., 1945).
Facultative parasites usually form trap structures in hyphae in presence of nematodes, specially the motile ones (Moosavi1 & Askary, 2015).Hohenbuehelia spp.produced traps in plates with and without nematodes (Figure 4), but none nematode were seen immobilized by traps.Askary (2015) reported many opinions about how trap are formed, most of them related to varied specific substances released by organs of nematode body as a possible stimuli to trap formation.There was one particular consideration on nutritional status, regarding to the confirmed influence caused by transference of mycelia from a richer agar medium to a poorer one, such as performed in the present study.Aparecido et al. (2008) noticed morphological changes induced by substrate changing within the same fungal isolate.It was unknown whether Hohenbuehelia species changed their mycelia pattern growth exclusively to capture nematodes so far, although our results show such changes also as consequence of mycelia transference from PDA to water-agar (2%) medium.
Hohenbuehelia paraguayensis and H. portegna preyed 34.96 and 17.17% of Panagrellus sp. at third day, respectively (Putzke et al., 2007) referent to an initial population of 500 nematodes, what means to say that both fungi preyed 174 and 85 individuals, respectively.However, in this study, H. portegna preyed 235 nematodes at third day, followed by H. paraguayensis and H. mastrucata with 183 and 109 nematodes preyed, respectively.These results matches to an affirmation of Chen and Dickson (2004) that pathogenicity variance among isolates is pretty normal all the more among different species.
Thorn and Barron (1986) proved that all Hohenbuehelia species correspond to Nematoctonus nematophagous species, however, they did not present a predation estimative by them.All isolates tested here controlled nematodes at some degree with direct hyphae penetration.
Panagrellus redivivus reproduces in fast rate, duplicating individuals in few days once females reach sexual maturity in 3 days, bearing approximately 10 to 40 juveniles at once (Sautter et al., 2007).Moreover, there was uneven reproduction in some quadrants causing high variability coefficient (Table 4).This reproduction could not be tracked up or marked on plate because nematodes moved a lot.
During control process, body penetration took some time to occur what classify the isolate as parasitic (Chen & Dickson, 2004) and as nematodes multiplied much, the predation score got high (Table 3) with predation surpassing 100%, since the more promising species preyed new borne juveniles too.
Capture leads to hypha penetration with progressive parasitism, but Nematoctonus genus does not form an infection bulb, specific structure of digestion process (Chen & Dickson, 2004).Anyways, body content was absorbed and total or partially disintegrated in 48 hours by all isolates.
According to Nordbring-Hertz et al. (2006), hyphae attachment on nematode cuticle depends on glycoproteins compatibility.Based on this affirmation, it is plausible to think that H. paraguayensis and H. mastrucata had higher compatibility, since their physical action over nematodes was more intense than for H. portegna.
Fungi predatory potential may be related to chemical specificity to nematode species and also among life cycle stages of them.This phenomenon happens to Paecilomyces lilacinus and Pochonia chlamydosporia, specialized in Meloidogyne spp.eggs parasitism, but with low predation on juveniles (Aït Hamza el al., 2017).Furthermore, Hohenbuehelia sp. had preference on P. redivivus than on Rhabditis (Thorn & Barron, 1986).
Gene expression determines the activity of some fungal species as Trichoderma sp. that produces chitin degrading enzyme while parasites nematodes eggs (Szabó et al., 2012).Therefore, even H. bullulifera and H. sp. did not showed high activity against juveniles of P. redivivus they could display another result against plant parasitic nematodes.Putzke et al. (2007) proved ability of H. paraguayensis and H. portegna on reducing gall formation of M. javanica in tomato roots in greenhouses trials, as well as, predation over Panagrellus spp.

Conclusions
None specie grew more influenced by nematodes movement under mycelium.Contrariwise, H. paraguayensis grew more in plates without nematodes.
The best predation performance was set by H. portegna and H. paraguayensis, followed by H. mastrucata, while H. sp. and H. bullulifera showed low potential as endoparasites agents.H. mastrucata had the best performance in growth speed, followed by H. portegna and H. paraguayensis.
Daily predation indexes revealed higher activity to H. portegna at 24 hours, H. mastrucata at 48 hours on and H. paraguayensis at 72 hours.
We did not report proportional relation between growth area and predation percentage to any specie, diclining our hyphotese.
This research indicates that Hohenbuehelia species produce substances with nematicidal properties that deserve future investigations.

Table 1 .
Growing of Hohenbuehelia spp.before nematodes addition