First report of edible mushroom Pleurotus ostreatus from India with potential to kill plant parasitic nematodes
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Edible fungus Pleurotus ostreatus was, isolated cultured and identified from the bark of Mangifera indica tree. The hyphae emerging from the fruiting body produces secretory cells with toxin droplets in water agar medium which immobilize the nematode and mostly enter from the mouth region. Earlier finding showed that trans-2-decenedioic acid toxin affects only saprophytic nematodes through the mouth part but similar observations in plant parasitic nematode Meloidogyne incognita suggested that there is no obstacle from stylet in paralysing and killing. From Indian subcontinent P. ostreatus was first time isolated, in perspective of plant parasitic nematode management in addition to mushroom production.
KeywordsMeloidogyne incognita Pleurotus ostreatus Mangifera indica Trans-2-decenedioic acid
Edible mushrooms, or wild edible fungi, have been collected and consumed by people from thousands of years and has been estimated that about 2000 species belonging to 31 genera are regarded as prime edible mushrooms Moore (2005). Among edible mushrooms, Pleurotus species is a common edible mushroom known for its characteristic oyster-shaped cap with an eccentric stalk attached to the pileus that opens up like an oyster shell during fruiting body formation and the presence of decurrent gills. They are white to various colour and enjoy worldwide distribution in nature from temperate to tropical regions, with the temperature range of 10–32 °C.
They also show carnivorous ability for killing nematodes by secreting toxin droplets and mycelia that contain cytoplasmic toxins (Barron and Thorn 1987; Thorn and Barron 1994; Tzean and Liou 1993). The toxin droplets produced by Pleurotus species was further characterized as fatty acid toxin Ostreatin having chemical composition of trans-2-decenedioic acid (Kwok et al. 1992).
The fruiting body of Pleurotus ostreatus (Jacq.) P. Kumm. was collected during rainy season from the bark of Mangifera indica in the premises of Banaras Hindu University, Varanasi, India. The fruiting body of mushrooms growing on the bark of trees were collected and brought to the laboratory and was further examined on the basis of colour, cross-section of fruiting bodies, examination of basidiospores, size, spore print and categorized as P. ostreatus on the basis of Pegler (1977) and Singer (1986) and on the basis of partial sequence of 18S rRNA blasted on NCBI Gene Bank data base with accession number MF939098. Small part from the fruiting body of the mushroom was taken by cutting it with sterilized blade and dipped in 0.01% HgCl2 solution for one to two second and consecutively washed three times with sterilized distilled water. After making it dry with sterilized filter paper, cut pieces of mushroom were kept in the plates poured with water agar medium.
For making pure culture population of saprophytic nematodes Panagrellus redivivus maintained on beef extract medium were used as bait from a nematode culture and placed on the water agar plates. Petri plates were incubated at 25 ± 1 °C and observations on interactions between nematode and fungal hyphae emerging from fruiting body were made under stereoscopic microscope. After 24–48 h, large number of saprophytic nematode were found to be killed with some of the mycelium arising from the pieces of mushroom kept in the sterilized Petri plate with the formation of toxin droplets.
The raised mycelium from the infection site bearing toxin droplets were picked up by sterilized needle under stereoscopic microscope and streaked on the plates poured with Potato dextrose agar medium and incubated at 25 ± 1 °C. Pure culture of the fungus was maintained by regular sub-culturing at the interval of 15 days.
For the evaluation of nematode killing ability of this P. ostreatus, twenty-five saprophytic nematodes were handpicked and placed on the growing cultures on corn meal agar medium (1:10). Observations on their attraction, trapping and killing were recorded with the formation of toxin droplets and secretory cells.
Lot of attraction and killing of saprophytic nematodes was observed (Fig. 1f, g) with the production of tiny secretory cells producing on the stalk bearing toxin droplets growing on the aerial hyphae or hyphae growing just above the surface of the medium with varying droplets size from 1.5 to 6.2 μm having stalk length 1.5–2.0 μm (Fig. 1g, h). The toxin droplets were visible on the vertical upward side of the hyphae growing on the surface of the medium while no droplets were seen on the horizontal position of the growing hyphae on the medium this might be due to the dissolving of toxin droplets at the site of hyphae touching directly on the medium. Secretory cells were observed most commonly on older hyphae than on the younger hyphae.
The plant parasitic nematode Meloidogyne incognita (Fig. 1e) and saprophytic nematodes P. redivivus (Fig. 1f) were found having larger attraction to the hyphae bearing secretory cells with droplets and most of them are immediately killed by mouth region just coming in contact with these droplets. After killing, P. ostreatus hyphae penetrate inside the nematode body from other sites also along with mouth region and proliferate inside the nematode body by disintegrating it completely to utilize its nutrients for their growth within 24 h of infection.
In earlier reports by the present research group, mechanism of several Pleurotus species was observed paralysing and killing the nematode in the same manner (Barron and Thorn 1987; Hibbett and Thorn 1994) as observed by us. In the present study, we have found that droplets were observed either on the aerial hyphae or vertical position of hyphae growing on the medium. No droplets were observed on the lateral side of hyphae growing on the medium. In most cases, it was observed that nematodes were found to be paralyzed and immobilized at the site of lateral position of hyphae that might be attributed to the content of toxin concentration emerging from the lateral hyphae dissolve at the site of medium which make the nematode paralyzed and subsequently secretory cells of hyphae penetrated inside the mouth region of nematode. This action happens so quickly that during observation only killed nematodes were seen no alive nematodes were observed entangling as observed struggling with trapping devices of other nematode trapping fungi.
This was first time noticed from several Pleurotus species killing saprophytic nematode in the same manner by Thorn and Barron (1984) and recognized these potent droplets as nematotoxin identified as trans-2-decenedioic acid. For studying the mechanism of this fungal- nematode interaction most of the workers have used saprophytic nematodes. In view of its practical utility, we have tried plant parasitic nematodes M. incognita to check whether these toxins are specific killing only saprophytic nematodes or stylet have any role for plant parasitic nematodes. During the study of Harposporium anguillulae, we have found that this fungus can parasitize only saprophytic nematode while stylet of the nematode act as a barrier for ingestion of its spore hence it was not found effective against stylet bearing plant parasitic nematodes which was also observed by Wang et al. (2007) while studying the infection process for several Harposporium species. Hence we have tested P. ostreatus against M. incognita and, M. graminicola and Heterodera avenae found very effective and killing in the similar fashion of saprophytic nematode.
To our knowledge, this is the first report of the P. ostreatus showing similar ability to attract or killing both type of saprophytic and plant parasitic nematode in vitro. Hence, the present study warrants a comprehensive work to utilize this fungus not only for the production of edible mushroom but also to restrict the plant parasitic nematodes population in the soil for causing disease in the field.
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