NEMATO-TOXIC POTENTIAL OF TRICHODERMA HARZIANUM AND T. VIRIDE EXTRA-CELLULAR METABOLITES AND ANNONA GLABRA CRUDE EXTRACTION ON MELOIDOGYNE INCOGNITA INFESTATION

Root-knot nematode, Meloidogyne incognita (Kofoid and White) is one of the most destructive plant parasitic nematode species in almost all agriculture and horticulture crops in Sri Lanka. Biologically sound controlling measures against this nematode species has been attempted from long past with ambiguous efficacy. This paper highlights the nemato-toxic potential of crude leaf extract of Annona glabra, and extracellular metabolites of Trichoderma harzianum and T. viride on root-knot nematode species, Meloidogyne incognita infesting in spinach plants under semi-field conditions. Results revealed that nematode infested plants treated with T. harzianum extract showed a significantly higher plant growth together with reduced root galling compared to that of T. viride and A. glabra crude extract. Both T. harzianum and T. viride treatments significantly increased the root growth of the nematode infested plants compared to that of A. glabra crude extract. Annona glabra crude leaf extract at the rate of 125 g/L and fungus mycelium of T. viride and T. harzianum at the rate of 140 mg/L and 100 mg/L *Corresponding author Email: deepika@kln.ac.lk; http://orcid.org/0000-0001-7727-1843 DOI: http:://doi.org/10.4038/josuk.v14i0.8028 This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution and reproduction in any medium provided the original work are properly credited. W.V. Lakmini and L.D. Amarasinghe 2 respectively resulted the highest nemato-toxic potential against M. incognita. This study concludes positive effect of antagonists tested against M. incognita and suggests that activity of lytic enzymes of T. harzianum and T. viride has enhanced the nemato-toxic effect.


INTRODUCTION
Meloidogyne incognita (Kofoid and White) Chitwood is a sedentary endoparasitic nematode species affecting many agricultural and horticultural crops (Sharon et al., 2001). The second stage juveniles of this nematode penetrate the epidermal cells closer to the root tip of the host plant. Once invaded, they locate the conductive tissues of the roots and commence feeding. Physiological changes due to the nematode feeding cause enlargement of the feeding cells and disrupt the functioning of conductive tissues. Formation of root galls is the characteristic symptom of root-knot nematode infestation while the nematode damage leads to a retarded plant growth, wilting during dry weather and reduction of crop (Taylor and Sasser, 1978).
Synthetic nematicides play a major role in controlling plant parasitic nematodes.
However, considerable negative impacts have been reported over their use. Therefore, as an alternative approach, several plant species and antagonistic fungi with strong nematicidal compounds have been assessed against root knot nematodes (Meyer et al., 2000;Khalil et al., 2012). It has been reported that crude leaf extract of Annona glabra (Family Annonaceae) contains Acetogenin, a high molecular weight compound having cytotoxic, anti-tumor, anti-parasitic, anti-fungal, anti-spasmodic, repellent, and insecticidal activities (Dang et al., 2011;Moghadamtousi et al., 2015;Alka et al., 2017).
In addition, previous studies also reported larvicidal effects A. glabra crude extract against mosquito species (Amarasinghe and Ranasinghe, 2017; Amarasinghe et al., an electric blender. Pulverized leaves, were subjected to soxhlet extraction using ethanol as the solvent. The thimble of the apparatus was filled with the powered leaf sample while ethanol was added to the round bottom flask of the apparatus. Extraction was carried out for 3½ hours maintaining the temperature at 78 °C. The resulted solution with extracted compounds were subjected to rotary evaporation at 40 °C until become a semi solid. The resultant crude extract was air dried for several hours until become a solid state and stored at 6 °C. Crude extract, 250 mg, was dissolved in 10 mL of deionized water and used as the stock solution (250 mg/L). Concentrations of 70, 125 and 170 mg/L of the crude were prepared by diluting the stock 30%, 50%, and 70% respectively for experimental use.

Preparation of liquid cultures of Trichoderma viride and T. harzianum
Laboratory maintained cultures of Trichoderma viride and T. harzianum were used for the study. A loop-full of the culture (5 mm diameter) from each species was inoculated on Potato Dextrose Agar (PDA) plates (5 x 5 x 1.5 cm) (n = 6) under aseptic condition (Amarasinghe & Madurusinghe 2012). Culture plates were maintained at room temperature, 28 ± 2℃, for six days and identified using morphological and reproductive characteristics (Watanabe, 2002;Anonymous, 2006). Pure cultures were maintained on PDA medium and stored in the incubator at 10 ℃. Fungal mycelium, of T. viride and T. harzianum, 20 mg each, were cut separately using an autoclaved cork-borer and was inoculated separately in fresh coconut water autoclaved at 15 psi (103 kPa) at 121 ℃ for 20 minutes, aseptically (Emerson and Mikunthan, 2015). Cultures were incubated for 30 days with occasional shaking at room temperature, 28 ± 2 ℃. The liquid cultures were filtered through a muslin cloth and the mycelial mass was discarded. The culture filtrates were used as the fungal stock solutions (200 mg/L). Concentrations, 60, 100, and 140 mg/L (30%, 50%, and 70%) were prepared by adding deionized water to Trichoderma stock solution and used for experimentations.

Potting of Spinach, Basella alba plants and inoculation
Transparent polythene bags measuring 9 cm diameter and 15cm height were filled with 250 g of pre examined nematode free soil and served as a pot. Three sets each of 40 pots were maintained for the experiments. Four week old Basella alba plants with six opened leaves were transplanted singly in each pot. Plants were given recommended NPK fertilizer at the planting and, watering done daily. Mature adult females of Meloidogyne incognita with egg masses were collected from field infested spinach plants and transferred individually into the rhizosphere of all the potted plants. These pots were kept in a screen house in semi field conditions for two weeks prior to the treatments.

Bio assay with A. glabra crude extract and extracts of Trichoderma
Annona glabra crude extract, 10 mL each from each concentration (30%, 50% and 70%) was applied into the rhizosphere of randomly selected ten potted plants each, previously inoculated with nematodes. Untreated control pots (n=10) received 10 mL water (4 T x 10 R). This was repeated for T. viride and T. harzianum. Pots were arranged in a randomized block design. Pots arrangement was rotated weekly to avoid the edge effect. Shoot height (cm), stem diameter (cm) at the level between first and the second leaf and total number of leaves were recorded and watered every other day. After six weeks, plants were carefully uprooted and root system was washed in running tap water; root length (cm) and galling in each root system were recorded.

Data analysis
The data analysis was performed using MINITAB 14 version. One-way ANOVA was performed to test whether there was a significant difference among the concentrations of treatments at 95% confidence interval. Anderson-Darling test was performed to determine the normality of the observed data. Tukey's pairwise comparison tests were carried out to determine whether there was a significant difference between test plants and controls.

RESULTS
Post treatment measurements, namely stem height, stem diameter, number of leaves and root length of treated and control plants infected with nematodes are given in Table 1. This shows that there is a significant difference in the number of leaves, stem height, and root length between treated and respective control plants (P = 0.001, F = 19.04; P = 0.074, F = 3.4; P=0.001 F=16.40 respectively) ( Table 1).