Management of Root-Knot Nematode, Meloidogyne incognita, in Tomato with Two Trichoderma Species

A population of root-knot nematode (Meloidogyne incognita) initially isolated from cucumber roots, identified on the basis of perineal pattern and maintained on the highly susceptible cultivar of tomato (money maker) was used in the assessment. The nematode was mass produced on tomato cv. Money maker and second stage juveniles were extracted from the infected roots for inoculation of plants (Mukhtar et al., 2017b).

For mass production of biocontrol agents Trichoderma harzianum and T. viride used in the experiment were obtained from the Institute of Agricultural Sciences, University of the Punjab, Lahore. Both the biocontrol fungi were mass produced on wheat grains. Wheat grains were chopped, soaked in water for 12 h, blotted dried and 250 g each was added in 500 ml flasks. The grains in flasks were double-autoclaved at 15 psi for about 30 min. The sterilized wheat grains in flasks were inoculated separately with pure cultures of each of the antagonistic fungi and incubated at 25±1°C for 15 days. The flasks were shaken at alternating days for uniform colonization of the fungi. The concentration of spores per gram of the grains was counted using haemocytometer after making spore suspensions in distilled water.

The soil (60% sand; 19% silt; 20% clay; 1% organic matter and pH 7.6) used in the experiment was sterilized with formalin, passed through a 3.5 mm mesh sieve to remove large stones and plant residues and added to pots.

Values are means of five replications. Means sharing common letters do not differ significantly according to Fisher’s Protected Least Significant Difference test (P > 0.05). T. h., T. harzianum; T. v., T. viride.

For abbreviations and statistical details, see Table I.

The comparative biocontrol potential of T. harzianum and T. viride against M. incognita was tested in pots. The biocontrol agents were each mixed with the formalin sterilized soil at the rates of 2×103, 4×103, 6×103, 8×103, or 1×104 cfu per g of soil. The treated soil (1 kg per pot) was then put in plastic pots. Three-week-old healthy seedlings of tomato cv. Money maker were transplanted individually in each pot. One week after transplantation, the plants were inoculated with approximately 2,000 freshly hatched second stage juveniles (J2s) of M. incognita. Nematode-inoculated plants without antagonists served as controls. There were five replications for each treatment. The pots were arranged in Completely Randomized Design in a greenhouse at 25±2°C for 7 weeks and watered as needed. After seven weeks, the plants were carefully removed from the pots. The shoots were severed from the roots. Shoot and root lengths were measured, and fresh root and shoot weights were determined. The galls and egg masses on each plant root system were counted under a stereomicroscope at 40×. For estimation of total nematode populations, eggs were extracted from the roots of individual plants and juveniles were extracted from the soil from each pot (Mukhtar et al., 2017b). The total number of eggs and nematodes in soil formed the total population. The reproductive factor was calculated by dividing the final population by the initial one. Percent reductions or increases in plant growth parameters and nematode infestations were calculated over controls as described by Kayani et al. (2018).

Completely randomized design was used in the experiment. All the data were subjected to analysis of variance using GenStat Package 2009 (12th edition) version 12.1.0.3278 (www.vsni.co.uk). Means were compared by Fisher’s Protected Least Significant Difference Test at 5%.

The application of T. harzianum and T. viride increased shoot weight and length and decreased root weight and length of tomato in a dose dependent manner (Tables I, II). On the other hand, both the antagonistic fungi caused significant reductions in number of galls, egg masses, eggs per egg mass and reproductive factors of M. incognita in a dose dependent manner (Tables III, IV). It was also observed that the reductions were slightly greater with T. harzianum than those with T. viride.

For abbreviations and statistical details, see Table I.

For abbreviations and statistical details, see Table I.

Trichoderma is a ubiquitous soil fungus which colonizes root surfaces and root cortices (Sharon et al., 2009). Several species of Trichoderma including T. harzianum, T. viride, T. atroviride, and T. asperellum, have provided excellent control of root-knot nematodes in previous studies (Sharon et al., 2007). Application of Trichoderma species resulted in reduced nematode galling and improved plant growth and tolerance. The highly branched conidiophores of Trichoderma produce conidia that can attach to different nematode stages. Conidial attachment and parasitism varies among fungal species and strains (Sharon et al., 2007). This process was often associated with the formation of fungal coiling and appressorium-like structures. T. harzianum colonizes isolated eggs and J2s of M. javanica (Sharon et al., 2007). Successful parasitism of the nematode by Trichoderma requires mechanisms to facilitate penetration of the nematode cuticles or eggshells. The involvement of lytic enzymes has long been suggested and demonstrated in Meloidogyne parasitism (Spiegel et al., 2005). Besides direct antagonism, other mechanisms involved in Meloidogyne control by Trichoderma spp. include production of fungal metabolites and induced resistance (Freitas et al., 1995; Goswami et al., 2008). In general, Trichoderma should be applied before planting to achieve maximum nematode control as good establishment of the fungus in plant rhizospheres seems to be important for nematode control.

The reason for increased plant growth, yield and other parameters observed here could be attributed to the release of growth promoting substances by bio-agents or by producing toxic metabolites which inhibit nematodes and exclude other deleterious microorganisms. The result obtained in current investigation uphold the results observed by Goswami et al. (2008) who observed increased growth and yield of tomato, soybean, tobacco and capsicum in pot and field experiments by the inoculation of Trichoderma spp. Reduction in nematode galls and egg masses might be due to high rhizosphere competency of bio-agents as they can easily colonize roots and may reduce feeding sites for nematodes. The reduction of root gall number may be due to the failure of majority of the juveniles to penetrate the host root. In the present study, the effectiveness of T. harzianum was slightly better than that of T. viride. This might be due to variations in genetics, pathogenic potential and the origin of the isolates.

It is concluded from the present evaluation that the indigenous isolates of T. harzianum and T. viride have the potential to control M. incognita and other species of root-knot nematode infecting vegetables.

Authors have declared no conflict of interest.