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Attraction of Root Knot Nematodes, Meloidogyne incognita, to Living Mycelium of Nematophagous Fungi

PJZ_50_6_2073-2078

 

 

Attraction of Root Knot Nematodes, Meloidogyne incognita, to Living Mycelium of Nematophagous Fungi

Manzoor Hussain*, Miloslav Zouhar and Pavel Ryšánek

Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamýcká 129, 16521, Prague, Czech Republic

ABSTRACT

This study was designed to detect the intensity of the attraction and repulsion of nematodes to several fungi. Of the 14 fungi tested, 11 attracted root-knot nematodes, one repelled nematodes, and two were neutral. The attraction intensity was directly proportional to time and based on the nutrients provided by the nematodes. In this experiment, the nematophagous fungi Arthrobotrys oligospora, A. superba, A. musiformis, Dactylella oviparasitica, Clonostachys rosea, Stropharia rugosoannulata, Lecanicillium muscarium, Trichoderma harzianum, T. viride, Pleurotus ostreatus, and Monacrosporium ellipsosporum demonstrated a prominent attraction intensity. The attraction intensity of all these fungi increased with time, while that of two fungi, Dactylaria gracilis and A. dactyloides, remained neutral throughout the experiment. Only the fungus species A. arthrobotryoides repelled the nematodes.


Article Information

Received 20 December 2017

Revised 13 March 2018

Accepted 11 April 2018

Available online 31 August 2018

Authors’ Contribution

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

Key words

Nematophagous fungi, Root-knot nematodes, Lecanicillium muscarium, Arthrobotrys arthrobotryoides, Attraction assay.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.6.2073.2078

* Corresponding author: hussain@af.czu.cz

0030-9923/2018/0006-2073 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan



Introduction

Parasitism or predation, mechanisms by which individuals from one species utilize or kill the individuals of another species as for sources of food (Abrams, 2000), exert substantial pressure on the prey of parasites or predators. To avoid being eaten, prey must evolve specific behaviors or strategies, such as camouflage, avoidance, trickery and threat displays, against their enemies (parasites or predators) in order to enhance their survival chances. Similarly, predators and parasites have also developed aggrandized predatory or parasitic competencies to secure the food requirements for their survival and reproduction. Therefore, the evolutionary race between prey and their predators or parasites is continuous and occurs on both sides to sustain the balance of ecosystems (Dawkins and Krebs, 1979; Anwar et al., 2007).

The mechanism used by plant parasitic nematodes to be attracted towards their hosts is still controversial among nematologists (Jones, 1960; Klinger, 1965), although several factors, such as temperature, electrical potential (Caveness and Panzer, 1960), carbon dioxide, various organic and inorganic substances (Croll, 1970; Jansson and Nordbring-Hertz, 1979) and root exudates (Bird, 1959; Van Gundy and Rackham, 1961), are thought to be involved. It is believed that nematode amphids have chemoreceptive organs that could be involved in locating hosts (Steiner, 1925). Some believe that nematodes locate hosts by moving at random in the rhizosphere and becoming trapped around root zones that are free of water and lacking the possibility of escape (Sandstedt and Schuster, 1962). Townshend (1964) reported that Aphelenchus avenae and Bursaphelenchus fungivorus were attracted to 57 of 59 plant pathogenic and saprophyte fungi. Nematodes directly oscillated to fungi colonies without any random movements. The carbon dioxide in the rhizosphere helps nematodes locate hosts, but nematode nervous and sensory systems are sufficiently developed to detect hosts from a certain distance (Chitwood and Chitwood, 1937). Nematophagous fungi are an important and prevalent group of soil microorganisms that facilitate the suppression of nematode populations (Nordbring-Hertz et al., 2011). These fungi are widely distributed in both terrestrial and aquatic ecosystems from the tropics to the Polar Regions (Pramer, 1964; Nordbring-Hertz et al., 2011). These fungi are categorized into four broad groups according to their modes of action. They are (i) nematode-trapping fungi using adhesive or mechanical hyphal networks, (ii) endoparasitic fungi using spores, (iii) egg-parasitic fungi invading nematode eggs with their sharp hyphal tips, and (iv) toxin-producing fungi paralyzing nematodes before penetration (Swe et al., 2011; Moosavi and Zare, 2012). All these fungi attract the nematodes towards them by diverting them from their path to a plant’s roots. Some fungi impede nematode movement by producing convoluted networks and prey devices, while others use toxic metabolites.

The objectives of this study were (i) to investigate the attraction of the root-knot nematode Meloidogyne incognita to the living mycelium of different types of nematophagous fungi and (ii) to determine the extent of nematode attraction or repulsion.

 

Materials and methods

Nematode culture

The Meloidogyne incognita population was prepared by inoculating a single egg mass onto the susceptible tomato variety Beril. Infected roots were brought into the lab, washed with running tap water, surface sterilized in 0.5% sodium hypochlorite for 2 min, washed three additional times with sterile water, and then placed in 10 ml tubes supplemented with the antibiotic streptomycin at a concentration of 1 g/l before incubation at 25°C. After 10 days, the contents of each tube were poured onto tissue in Baermann funnels, and after 24 h, the J2 that hatched were collected, counted and used for tests (Hussain et al., 2016a, b, 2017a, 2018).

Fungi culture

Fungi already maintained on potato dextrose agar (PDA) at 20°C were cultured on corn meal agar (Difco), diluted 10-fold (CMA 1:10) and subcultured once a month. During the attraction/repulsion tests, fungi were allowed to grow almost to the edges of the plates, which took 1 to 3 weeks, depending on the species. The growth of some fungi was relatively slower than the growth of others, and some fungi were observed to produce trap devices after 3 weeks (Jansson and Nordbring-Hertz, 1979).

Attraction assay

The attraction and repulsion intensities of the fungi were determined based on the methods described by Jansson and Nordbring-Hertz (1979). Discs of fungal colonies (1 cm diameter) were cut with a cork borer from the edge of a colony growing on CMA 1:10 (0.2% agar, 2 mm thickness) and placed in two quadrants (I, III) on fresh CMA 1:10 plates 1 cm from the edge of the plate. In the other two quadrants (II, IV), control discs of media CMA 1:10 without fungi were placed as described in Figure 1. The plates were incubated for 24 h to allow the successful development of the diffusion of concentrated substance gradients.

A distilled water drop containing the nematode suspension (with approximately 100 juveniles) was placed in the middle of the plate. The nematodes present in, on and under the discs were counted using a stereomicroscope after 24 h. The average value was taken from quadrants I and III and similarly from quadrants II and IV. To avoid nematodes amassing in the middle of the plates, they were dried for at least 2 or 3 days before being observed under the microscope. Amassing could happen if a large quantity of nematodes were used in the plate. The number of traps (constricting rings) induced in response to the nematodes was recorded after 24 h with a microscope at 100× magnification. The trap devices were counted around the fungus base and the middle and sides of the petri plates with 9 inches diameter.

Dilute corn meal agar facilitated microscopic observations of both the mycelium and nematodes by furnishing a thin mycelial mat. The Petri plates were incubated at 28±1°C with five replications.


 

Attraction intensity assay

The same procedure was adopted as described above, except that the number of nematodes aggregated under each disc was counted hourly. To ensure that the nematodes were not captured by trap-bearing nematophagous fungi, the assay was restricted to 7 h (Jansson and Nordbring-Hertz, 1979).

Ultraviolet (UV) irradiation

The plates were irradiated with UV light (254 nm, 2 W, 25 cm, 20 min) to neutralize the effects of nematophagous fungi. Immediately after the UV treatment, nematodes were introduced to the plates to complete the assay (Jansson and Nordbring-Hertz, 1979).

Data analysis

The lab experiment was conducted two times with five replications. Data were pooled and subjected to ANOVA tests; means were partitioned by the least significant difference (LSD) test by using the software Statistics 8.1.

 

Table I.- Attraction of the root-knot nematodes, Meloidogyne incognita to several fungi on CMA 1:10 after 24 h.

Nematophagous fungi

Formation of trap devices

Attraction/ repulsion*

Attraction/ repulsion after UV treatment λ

A. oligospora

+

+

0

A. superba

+

0

A. musiformis

+

0

A. dactyloides

+

0

0

A. arthrobotryoides

+

C. rosea

­−

+

+

S. rugosoannulata

+

+

ND

L. muscarium

+

0

T. harzianum

+

0

T. viride

+

0

P. ostreatus

+

ND

M. ellipsosporum

+

+

+

D. oviparasitica

+

+

0

D. gracilis

+

0

0

ND, not determined; * +, attraction; −, repulsion; 0, neither attraction nor repulsion. All values significant at P < 0.001 at ten replications. λ, attraction/repulsion of fungi killed by UV irradiation prior to addition of nematodes.

 

Results

Attraction assay

Based on results of the attraction assay, the fungi can be divided into three groups. The first group of fungi attracted nematodes, the second did not show any activity during the experiment, and the third proved to be repellant to nematodes. Moreover, the highest intensity of the attraction of nematodes to the fungi was found in two fungi species, L. muscarium and S. rugusoannulata, which attracted over 80% of the nematodes in both quadrants (I, III) after 24 h. Out of the fourteen fungi species, eleven attracted nematodes, and two had an undetermined effect because they did not demonstrate any level of attraction. The fungus species A. arthrobotryoides was repellant to nematodes. Based on the trap devices observed after 24 h, the fungi with the highest attraction capability were S. rugusoannulata, A. oligospora, A. dactyloides, and M. ellipsosporum. The second highest capability was observed for Arthrobotrys oligospora and Trichoderma harzianum, which attracted more than 70% of the nematodes from both quadrants (Table I). The lowest attraction capability, irrespective of trap devices, was recorded for Monacrosporium ellipsosporum, as shown in Table II. Furthermore, Clonostachys rosea, A. superba, and Monacrosporium ellipsosporum were observed to be slow-growing species on corn meal agar.

 

Table II.- Attraction of root knot nematodes, Meloidogyne incognita to different fungi after 24 h.

Nematophagous fungi

Fungal treatedQuadrant I, III (Mean)

Control Quadrant II, IV (Mean)

L. muscarium

41a

3gh

S. rugosoannulata

40a

1hi

A. oligospora

37b

3gh

T. harzianum

36b

4fg

P. ostreatus

28c

4fg

T. viride

28c

7f

C. rosea

27c

1hi

A. musiformis

26c

4fg

A. superba

22d

6f

D. oviparasitica

22d

7f

M. ellipsosporum

12e

1hi

A. arthrobotryoides

0i

6f

D. gracilis

0i

7f

A. dactyloides

0i

6i

All values significant at P < 0.001 at ten replications.

 

Attraction intensity assay

The attraction intensity assay was performed for all fungi. The results showed that with increasing time, the attraction intensity of all fungi also increased, except for three species (A. arthrobotryoides, D. gracilis, A. dactyloides), which were declared as neutral during the experiment. The maximum increase in attraction over time was observed for the species L. muscarium, S. rugusoannulata and Pleurotus ostreatus, while the minimum value was found for M. ellipsosporum, D. oviparasitica and A. superba, as shown in Figure 2. The rest of the fungi showed moderate attraction intensity over time (Fig. 2).

Ultraviolet (UV) irradiation

To investigate the attraction/repulsion impression of nematophagous fungi, a UV irradiation experiment was employed. The results showed that after fungi were irradiated with UV light, they no longer attracted nematodes, except for the species M. ellipsosporum, which maintained its attraction capability. The activity of the neutral fungi remained constant.


 

Discussion

The study not only determined the attraction and repulsion of nematodes towards nematophagous fungi but also facilitated a comparison of the effects among fungi species. We observed that most of the fungi were nematophagous, except for a few that were neutral during the experiments. The attraction/repulsion intensity of the neutral fungi showed that they were non-nematophagous. We observed that fungi were capable of attracting nematodes irrespective of trap formation. According to our study, trap devices are not important to attract the nematode (Field and Webster, 1977; Jansson and Nordbring-Hertz, 1979) but are key weapons to capture and infect nematodes when fungi switch from the saprophytic to predacious phase (Yang et al., 2011). Moreover, it has been revealed that A. arthrobotryoides produces trap devices but repels the nematodes (Jansson and Nordbring-Hertz, 1979). In this study, A. dactyloides and D. gracilis produced trap devices but did not attract the nematodes. We assume that for these kinds of fungi, nematodes must be near the traps to be parasitized, and the fungi do not produce any attractant or stimulant enabling them to attract the nematodes. On the other hand, there is a small polypeptide or amino acid called “nemin” present in most nematodes (Pramer and Kuyama, 1963) that may also be responsible for the induction of trap formation. Our results contradict those of the studies conducted by Singh et al. (2007) reporting that A. dactyloides was the most efficient nematophagous fungi during in vitro studies by the induction of constricting rings against Meloidogyne graminicola. Our investigation is more closely related to attraction phenomena than parasitism. Certainly, when the nematodes get closer to the constricting rings, they could efficiently be captured and consumed by A. dactyloides.

The fungi L. muscarium, S. rugosoannulata and A. oligospora showed the maximum intensity of attraction to nematodes, which seems to suggest that they are better parasites of nematodes (Hussain et al., 2017b, c, d, e, f). These fungi may contain signaling substances that attract the nematodes towards them. This study also observed that almost all fungi lost their attraction capabilities after they were killed with UV light, except for C. rosea and M. ellipsosporum. This result suggests that there are volatile substances present that have the ability to attract nematodes (Jansson and Nordbring-Hertz, 1979). The measurement of attraction intensity over time also proved that with the increase in time, attraction increases. According to Hsueh et al. (2017), if not lured by volatile compounds, nematodes could escape from A. oligospora before trap devices are fully functional after 12 h.

 

Conclusion

Our data shows that the nematodes are attracted to nematophagous fungi during the period of trap formation and later captured by the functional traps.

 

Acknowledgements

We are grateful to the Czech University of Life Sciences, Prague, for providing funds to conduct part of this research under CIGA No. 20152016 and Ministry of Agriculture No. QJ1310226.

 

Statement of conflict of interest

All authors declare that there is no conflict of interest in this study.

 

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