Effect of fungal inoculums
Plant parasitic nematodes cause great economic losses to agricultural crops worldwide. They along, with their hosts, are not isolated in the ecological system, but are strongly influenced by antagonists, parasites and pathogens. Though pesticides appear to be the most economical and efficacious means of controlling plant pathogens, toxicological, environmental and sociological concerns have led to drastic reductions in the availability of efficient commercial nematicides. These restrictions have forced farmers to look for an integral system that makes use of other means of disease control. Species of spiral nematodes, Helicotylenchus and Scutellonema, were among the most abundant plant parasitic nematodes of the mulberry plant. Eco-friendly control of the parasitic nematodes could be achieved by means of endoparasitic fungi (like Hirsutella, Meria, Nematophthora and Nematoctonus), trapping fungi (like Arthrobotrys and Duddingtonia) or parasitic fungi (like Paeceilomyces lilacinus). During the course of this present work, Trichoderma Pers. Ex. Fr. was found to be one of the most effective fungi in controlling the eggs and J2 of Meloidogyne javanica. The present study outlines the comparative efficacy of five Trichoderma species (T. viride, T. harzianum, T. longibrachiatum, T. koningii and T. hamatum) against Helicotylenchus sp. and Scutellonema sp. The study also outlines the effect of Trichoderma viride Persoon on Scutellonema spp. and Helicotylenchus sp., effect of Trichoderma harzianum Raifae on Scutellonema sp. and Helicotylenchus sp., effect of Trichoderma longibrachiatum Rifai on Scutellonema sp. and Helicotylenchus sp., effect of Trichoderma koningii Oudeom on Scutellonema sp. and Helicotylenchus sp., and lastly effect of Trichoderma hamatum (Bonord) Bainier on Scutellonema sp. and Helicotylenchus sp.
- Plant parasitic nematodes
- mulberry plant
- antagonistic Trichoderma spp
Plant parasitic nematodes cause great economic losses to agricultural crops worldwide. They along, with their hosts, are not isolated in the ecological system, but are strongly influenced by antagonists, parasites and pathogens. Though pesticides appear to be the most economical and efficacious means of controlling plant pathogens, toxicological, environmental and sociological concerns have led to drastic reductions in the availability of efficient commercial nematicides. These restrictions have forced farmers to look for an integral system that makes use of other means of disease control. This imperative approach involves a mixture of agro-technical, biological, chemical and genetic (breeding) means of plant disease control [20, 24, 36]. Species of spiral nematodes,
Eco-friendly control of the parasitic nematodes could be achieved by means of endoparasitic fungi (like
During the course of this present work,
2. Materials and Methods
2.1. Extraction of nematodes
Soil samples from around the rhizospheric regions of mulberry plants were collected and processed through Cobb’s sieving and decanting method followed by Baerman’s funnel technique . The nematodes were observed under stereoscopic microscope and were counted using a Syracuse counting disc.
2.2. Isolation and enumeration of
Trichodermasp. from soil
The fungi were isolated through the serial soil dilution plate method . Then, 10 g of oven dried fungi was added to a sterile Erlenmeyer flask with 90 ml sterile water, and the mixture was stirred with a magnetic stirrer for 20-30 minutes. A blender was used for blending the samples. While the suspension was in motion, 10 ml of solution was taken and added to 90 ml sterile water in a screw-cap flask or medicine bottle. It was shaken for one minute and 10 ml of the suspension was transferred to a 90 ml sterile water blank. The process was repeated until the desired dilution was obtained. Ten millilitres of soil solution was pipetted and mixed with 90 ml distilled water and marked to 10-3. From 10-2 and 10-3 test tubes about 5 ml solution was added to culture media contained in four petri dishes (two of each) and kept at laminar flow for 3-4 days.
To facilitate uniform spreading of the suspension over Czapek Dox agar surface at pH 5.5, the plate was placed on a turntable and the suspension spread with a flamed L-shaped rod with one hand, while rotating the turntable with the other. To obtain distinct colonies, plates were prepared 2-3 days before use or placed for a few hours at 35 to 40° C after pouring to ensure a dry agar when the suspension was added. A water film on the freshly poured plates caused excessive spreading of organisms. The plate was incubated for a few days at 24-30° C and colony counted.
The composition of the culture media was as follows:
Sodium nitrate (Na2 NO3) - 1.0 g
Magnesium sulphate (Mg SO4⋅7 H2O) - 0.5 g
Potassium chloride (K Cl) - 0.25 g
Potassium dihydrogen phosphate (KH2PO4) - 0.5 g
Ferrous sulphate (FeSO4.7 H2O) - 0.5 g
Sucrose - 15.0 g
Agar agar - 10.0 g
Distilled water – 500 ml
A broth media was made by mixing together the substances excepting the agar. It was kept for 24 hours to dissolve the substances completely. Five to six drops of the broth were removed with a dropper into different autoclaved cavity blocks.
2.3. Inoculation of nematode and fungi
Five to six drops of the broth were removed with a dropper into different autoclaved cavity blocks. Then, 0.1 ml of selected
3. Results and Discussion
The fungus attacked the nematodes though the production of conidia, sticky spores and mycelia, which on contact adhere to the cuticle and germinate, forming germ tubes that penetrated the nematodes. Then, they extended their hyphae inside the nematodes after penetration of the cuticle by conidia formation. These hyphae multiplied profusely. They inactivated the parasitic nematodes and immobilized them due to production of certain antibiotics and compounds. Observations of the immobility and parasitism of the nematodes due to the fungi were made every 12 hours. Each observation was replicated three times and the results are represented in tables 1–10. Photographs with graphs of parasitism are also provided in figures 1-32.
After 12 hours of inoculation, the fungus produced mycelium and conidia. The highest immobility was found at 108 hours of inoculation. The spores attached at the middle and anterior end of the body, and made the nematode immobile in the case of
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3.2. Effect of
Trichoderma harzianum Raifaeon Scutellonemasp. and Helicotylenchussp. (table 3 and 4)
There was no infection after 24 hours of inoculation, but 3 % of the nematodes were immobile. Infection and parasitism of the nematode occurred after 48 hours of inoculation. The highest immobility was found at 108 hours of inoculation. Many mycelia grew over the body of the nematode. The conidiophores were seen to be multiple-branched, forming loose tufts which arose in distinct and continuous ring-like zones. The main branches, mostly in groups of two or three, stood at right angles, and the length increased with the distance from the tip of the main branch, giving a conical or pyramidal appearance. The body cuticle of the nematode was suppressed. The mycelia tip ran parallel to the nematode. There was rapid and excessive coiling on the target host. The mycelium coiled with its constricting networks of loops at the anterior region of the body and the head region, making constrictions that might be due to the sucking of body contents. After 96 hours of inoculation, there was complete immobilization of the nematodes. In the case of
3.3. Effect of
Trichoderma longibrachiatum Rifaion Scutellonemasp. and Helicotylenchussp. (table 5 and 6)
After 12 hours of inoculation, 4 % of the total nematode population was found to be immobile, with the highest immobility at 108 hours of inoculation. Infection started before 20 hours of inoculation. Hyphae of the fungus strain formed an appressorium-like structure in close contact with the nematode. They produced penetration holes in the cuticle of the nematode. The penetrated cuticle rapidly lost turgor and collapsed. At contact with the nematode cuticle, the hyphae branched dichotomously at the tip. The hyphae were not observed to coil around the nematode cuticle, and instead grew along the cuticle. However, penetration was not evident. Despite the absence of visible penetration, the nematode cuticle lost turgor pressure, wrinkled and collapsed. Finally, both the cuticle and body content of the nematode completely disintegrated. In the case of
There was no effect during the first 12 hours of inoculation in the case of
3.5. Effect of
Trichoderma hamatum(Bonord) Bainier on Scutellonemasp. and Helicotylenchussp. (table 9 and 10)
There was no effect on the nematode
Several possible mechanisms have been suggested to be involved in
The tested species of
Using beneficial fungi for control of plant disease is a useful and acceptable method for farmers. As such, the fungal microbe