Abstract: Antagonistic effect of five species of Pleurotus, including P. ostreatus, P. sajor-caju, P. cornucopiae, P. florida and P. eryngii on second stage juveniles (J2) of Meloidogyne javanica were studied, in vitro. On water agar, all of the species tested produced tiny droplets of toxin. Nematodes touching such droplets showed a sudden response and became colonized by the fungi after 24-48 h. Filtrates of the tested fungi grown in malt extract broth were toxic on the nematodes but this toxicity varied between species. Culture filtrates of P. ostreatus showed the highest nematicidal activity toward M. javanica J2 and the lowest toxic effect was observed in filtrates of P. eryngii. A linear relationship was proved between increasing toxin concentration and the percentage of dead nematodes.
INTRODUCTION
Root-knot nematodes (Meloidogyne spp.) are one of the most economically important pest causing sever damages to a wide variety of crops particularly to tomato (Siddiqui and Shaukat, 2003). Their wide host ranges (Jepson, 1987) made them difficult to be controlled by rotation and resistant cultivars have variable value because of the occurrence of virulent races and species mixtures (Roberts, 1992). They also have rapid rates of multiplication on good hosts, further increasing the difficulty of preventing crop damage as small populations increase and become damaging during one growing season (Trudgill, et al., 2000). Resistance genes effective against Meloidogyne spp. Appear not to have resulted from coevolution (Trudgill and Block, 2001). Existing management procedures could be enhance by the development of biocontrol strategies (Siddiqui and Shaukat, 2003). Nematicides are used to control various species of root-knot nematodes in developed agriculture but are mostly inappropriate for subsistence farmers in developing countries. Consequently, there is a great need to increase the control options for managing root-knot nematodes and biological control has been an active area of research.
At least 10 species of gilled fungi belonging to the genera Hohenbuehelia, Pleurotus and Resupinatus can attack nematodes by adhesion or toxins (Tzen and Liou, 1993). Thorn and Barron (1984) found that when nematodes came in contact with the hyphae of Pleurotus, they became inactive very quickly and were subsequently colonized and digested by the fungus. Pleurotus species display a kind of nematode capture that appears to be unique to this genus. They produce tiny appendages on the vegetative hyphae and these secrete droplets of a potent toxin (Thorn and Barron, 1984; Barron and Thorn, 1987; Peterson, 1993; Thorn and Tsuneda 1993). The toxin produced by Pleurotus ostreatus has been identified as trans-2-decenedioic acid (Kwock, et al., 1992). Barron and Thorn (1987) reported that rhabditid nematodes touching such droplets, showed a sudden response, the head region shrinked, hyphae attracted to the body orifices and homed in on the head. This mode of nematode attack has been observed in P. cornucopiae, P. cystidiosus, P. levis, P. pulmonarius (Thorn and Barron, 1984; Thorn and Tsuneda 1993), P. tuberregium, P. dryinus, P. euosmus, P. eryngii (Hibbet and Thorn, 1994) and P. sajor-caju (Sharma, 1994). The toxin produced by P. cystidiosus is present either at much lower concentration or is structurally different from that of P. ostreatus (Barron and Thorn, 1987). Aphelenchoides composticola, one of the most damaging mushroom nematodes, immobilized in P. sajor-caju culture filtrates within 2 to 4 h. of the exposure (Sharma, 1994).
It was suggested (Barron and Dierkes, 1977; Thorn and Barron, 1984) that nematodes may be an important nitrogen supplement for fungi, especially in wood substrates where the C:N ratio is very high and that the predatory capability of wood decay fungi has evolved to satisfy their nitrogen requirement. The host range for Pleurotus has never been fully established. Toxin droplets can be instrumental, not only in supplying a nitrogen source to Pleurotus, but can also function as an antifeedant that protect hyphae from fungus feeding microfauna (Barron, 2003). In this study, the nematoxic effects of some species of Pleurotus has been tested in vitro.
MATERIALS AND METHODS
The present investigation has been performed in the Department of Plant Pathology Tarbiat Moddares University in the year 2003-2004. Single-egg-mass cultures of Meloidogyne javanica were maintained on tomato (Lycopersicon esculentum) cultivar. Early Urbana in an autoclaved (140°C, 40 min) sandy soil in 18 cm-diameter pots in a glasshouse (14 h. light and 10 hrs. dark daily; temperature 24-30°C and RH 75-85%). Eggs were extracted by the NaOCl technique (Hussey and Baker, 1973). Second stage juveniles (J2s) were set to hatch from the eggs spread on a 30 μm sieve (Baker, 1985), collected daily and stored at 15°C. Juveniles aged less than 3 days were used in all experiments.
Behavior of M. javanica juveniles in Pleurotus cultures:
Pure cultures of P. cornucopiae (ITCC No: 1724), P. eryngii
(ITCC No: 3046), P. ostreatus (ITCC No: 2572), P. sajor-caju (ITCC
No: 1725) and P. florida (ITCC No: 3308) were collected from Indian Type
Culture Collection (ITCC) and maintained on potato dextrose agar (PDA) and transferred
or subcultured weekly. Agar disks (5 mm) diameter were cut from 6-day-old
cultures and each disk was placed at the center of water agar (2% w/v) plates
and grown for 5-7 days at 24°C. By this time, the fungi had radiated a distance
of 2-4 cm from the disks. Twenty active J2s of M. javanica previously
surface sterilized in 5% streptomycin solution (De Ley and M-Ocampo, 2004) were
placed at the edge of the colonies. Periodic observations were made under a
stereoscopic microscope.
Viability of the juveniles in Pleurotus culture filtrates:
Erlenmeyer flasks (500 mL) containing 300 mL of Malt Extract Broth (MEB),
were inoculated with a 1 cmblock from a 7 days old fungal colonies growing on
PDA. The fungi were cultured for three weeks on a rotary shaker at a low speed
of forward and backward motion (40-50/min), at room temperature (23-24°C).
The filtrates were first passed through a layer of filter paper (whatman cat.
No: 1001110) and then through a 0.45 μm filter. The filtrates were used
at concentrations of the original preparation (1x) and diluted 5x or 25x (Sharma,
1994).
A suspension of 100 J2 mL-1 of sterile water was prepared. About 50 surface sterilized J2 in 0.5 mL of water were mixed with 5 mL of the culture filtrates in a Petri-dish of 4cm dia., fresh malt extract broth or sterile water. Four replicates were tested per treatment. Viability of the J2s was determined after 4, 8 and 24 h and the percentage of paralyzed J2 was calculated (Moje, 1959). Juveniles that did not move even after touching the tail were considered to be dead. So, after the incubation period, culture filtrates were replaced with tap water and mortality of the juveniles were checked after 20-24 h.
The experiment was repeated. A completely randomized design was used. Data were subjected to analysis of variance (ANOVA) and means were compared with the Least Significant Differences (LSD) test (p = 0.05).
RESULTS
Behavior of M. javanica juveniles in Pleurotus cultures:
Secretory processes were observed on aerial hyphae of all tested species.
From the top, only the round outlines of the toxin droplets were visible (Fig.
1A). Viewed from the side, the secretions were seen as a slender stalk,
leading to a rounded head (Fig. 1B). Nematodes moved without
any harm over the young hyphae at the periphery of the colony, where there were
no toxin droplets were absent. After touching the droplets on the older parts
of the mat, they usually recoiled immediately. By one or more contact, nematodes
became inactive and after 24-48 h. it was noticed that the hyphae of the fungi
had grown toward the nematodes and penetrating them through body orifices especially
through their mouth (Fig. 1C and D). The
body contents were digested within 2-3 days (Fig. 1E). All
species tested caused similar symptoms on nematodes, but the length of exposure
time and the number of contacts required for appearance of these different symptoms
were varied between fungi tested. the J2s showed severe reactions after contact
with toxic droplets of P. ostreatus and inactivated quickly. Similar
symptoms could be seen after prolonged exposure to a large number of droplets
of P. eryngii.
Effect of Pleurotus culture filtrates on viability of M. javanica J2s: The effect of culture filtrates on J2s viability varied between the fungal species.
| Fig. 1: | Nematode trapping structure of Pleurotus ostreatus and nematodes on agar cultures of Pleurotus ostreatus. A. Toxin droplets on aerial hyphae, viewed without coverslip. B. side view of toxin droplet on stalked appendage, C. viewed with coverslip. D. Hyphae of Pleurotus ostreatus homing in on the mouth of nematode on water agar. E. Meloidogyne javanica J2 colonized by hyphae of Pleurotus ostreatus. |
Culture filtrates of P. ostreatus and P. sajor-caju, showed the highest nematicidal activity toward M. javanica J2s (Table 1). Culture filtrate of P. ostreatus in 1x malt extract broth; paralyzed 78% of J2s after an exposure period of 4 h and all of them after 24 h. A similar toxicity was observed in culture filtrates of P. sajor-caju. Culture filtrates of P. cornucopiae and P. florida in 1x, exhibited similar toxic activities to J2s and paralyzed 80 and 76% of J2s, after 24 h respectively. Culture filtrate of P. eryngii showed the lowest toxicity on J2s and there was not significant difference between 25x filtrate dilution of P. eryngii and controls (Table 1). A linear relationship was proved between increasing concentration and the percentage of paralyzed nematodes. Repeated experiment showed similar results (Table 2).
| Table 1: | Percentage of second-stage juveniles of Meloidogyne javanica paralyzed after a period of incubation in filtrates of fungal cultures in malt extract broth |
| Table 2: | Percentage of second-stage juveniles of Meloidogyne javanica paralyzed after a period of incubation in filtrates of fungal cultures in malt extract broth |
DISCUSSION
In our study, all of the tested Pleurotus species showed toxic effects on M. javanica juveniles. Our observations proved the previous reports (Hibbet and Thorn, 1994) about toxic droplets. The nematodes did not show severe symptoms such as shrinkage of the head region reported previously for rhabditd nematodes (Barron and Thorn, 1987). This may be due to the structure of the buccal cavity of plant parasitic nematodes that is different from free living or bacteriophagous nematodes. As reported (Barron and Thorn, 1987), there was no relationship between the droplet density and nematicidal activity of the species. Thus the concentration of the toxin produced by the species may be different or the toxins may be structurally different in various species.
Toxin production in liquid media is influenced by a number of factors such as culture medium, aeration of media and pH (Chen, et al., 2000). Variation of toxin production among strains within species has also been observed (Hallman and Sikora, 1996). Because toxin production of fungi was influenced by culture media (Chen et al., 2000), the effect of toxins on nematodes may not become clear until toxin production by fungi is evaluated in situ. The toxin production in soil or on a root may be different from that in culture media. Further experiments should be performed to determine whether and under which conditions these fungi can produce toxins in large and effective amounts.