Effect of Paecilomyces lilacinus on tomato plants and the management of root knot nematodes

Effect of Paecilomyces lilacinus on tomato plant growth and the management of root knot nematodes in tomato was studied. The research work was conducted in Microbiology & Bio-control Laboratory, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh and in Net-house of Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, during the period from July, 2016 to October, 2017. In net-house pot culture experiment, four different treatments were used viz., T1: Inoculation of egg masses (10 egg masses/plant) of Nematodes (Meloidogyne spp.), T2: Application of P. lilacinus, T3: Application of egg masses of Nematodes and P. lilacinus simultaneously, and T4: Control (non-treated). Spore suspension (10×10 Conc.) of P. lilacinus was mixed with the soil before transplantation and Meloidogyne spp. was inoculated on three days after transplantation. Application of P. lilacinus in soil enhanced the plant growth parameters of tomato plants. Inoculation of Meloidogyne spp. reduced plant growth and the reduction was increased with the increase of inoculum density of Meloidogyne spp. Maximum plant growth reduction was recorded when Meloidogyne spp. was inoculated alone. The maximum plant growth was recorded in case of application of P. lilacinus to soil. A high percentage (85%) of egg masses of Meloidogyne spp. was infected by P. lilacinus when applied together. Copyright: ©2019 by authors and BAURES. This work is licensed under the Creative Commons Attribution International License (CC By 4.0).


Introduction
The tomato belongs to the family Solanaceae and the species Solanum lycopersicum is a premier vegetable in Bangladesh and are widely cultivated (Lucioli et al., 2014).Regular outbreaks of root-knot nematode disease caused by Meloidogyne spp.have occurred in recent years, impacting considerably on both tomato crop yield and quality, and are increasing problem in global tomato production (Wesemael et. al., 2011;Seid et. al., 2015).Management of Root knot nematodes is difficult due to its wide host range.Once the root knot nematode establishes a feeding site, a group of cells known as giant cells, it remains permanently attached at that location within the plant root.An increase in production of plant growth regulators occurs due to esophageal gland secretions from the nematodes, which cause an increase in cell size and division, resulting in gall formation.Nutrient deficiency symptoms may also appear on plants; chlorosis and stunting frequently occur in root knot nematode infested fields.If high populations of root knot nematode occur early in the growing season, the host plant can be killed (Rahim et al. 2016).Among more than 80 species of the genus Meloidogyne, four important species viz., M. incognita, M. Javanica, M. arenaria and M. hapla were responsible for at least 90% of all damages as root parasites (Castagnone-Sereno, 2002).Tomato is highly susceptible to infection by the species M. incognita and M. javanica (Khan et al. 1984).Some chemical nematicides (insecticides) are reported to control effectively the root-knot nematodes of tomato.But for killing the pests, nematicide exerts adverse effects on human beings, livestock and other living things, which come in contact directly or indirectly.In this condition, there is a need to use bio-pesticides that are pest specific, nontoxic to humans, less expensive, and safe for the environment.Environmental side effect associated with chemical control and the loss of methyl bromide as a multipurpose soil fumigant have spurred research into nematode control alternatives (Nico et al., 2004).Researchers all over the world are engaged in standardizing nematode management strategies by following non-chemical and eco-friendly approaches such as biological control agents to stabilize crop production (Sumathi et al., 2006).Several attempts have been made to use antagonistic fungi to control root-knot nematodes.
Biological control is gaining popularity in nematode control, predominantly utilizing the microorganism groups like the fungi and bacteria already present in the soil biota (Crawford and Clardy, 2011).Many microorganisms reveal the capacity to parasitize the egg and juvenile forms and sometimes even adult nematodes.Paecilomyces lilacinus is a soil-inhabiting fungus with a wide range of activity against the most important plant parasitic nematodes (Vasanthi & Kumaraswamy, 1999;Brand et. al., 2010).Siddiqui et al. (2000) reported that P. lilacinus significantly reduced Meloidogyne spp.infection on tomato.Thus, the present investigation was designed to evaluate the effect of P. lilacinus on tomato plants and the management of tomato root knot caused by nematodes.(Monjil and Ahmed, 2017).Purified cultures (from seven days old fungal culture) of the isolated fungi were examined microscopically in order to identify the strain on the basis of their morphological traits and cultural characteristics of the fungi such as mycelium growth, colony texture, spores production and other characteristics (Abubakar et al., 2005;Ahmad and Jairajpuri, 1993).

Collection of root knot diseased materials
Possible nematode infested tomato plants were selected by observing above ground symptoms.Selected plants were uprooted by digging with spade and sickle.Caution was taken to get intact root system without leaving any root parts or gall produced by nematodes.Nematode eggs were separated from previously collected heavily galled (knotted) tomato roots in the laboratory.Only well developed light brown and live egg masses were selected for the research work.Nematode identification was performed by using photographic microscope (Carl Zeiss Microscope GmbH, Model Stemi 508), at Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh.Melodogyne spp.were identified by observing their identifying characters as described by Eisenback and Hirschmann (1981).

Net-house experiment
The soil of the experiment was collected and sun dried for two days.Then the soil was grounded, and large particles and plant debris were removed.Compost was collected from the BAU-farm, Bangladesh Agricultural University, Mymensingh.Soil and compost were composited and mixed well in a ratio of 4:2.The mixture of soil sterilized with formalin 5% solution @ 200 ml /cft soil.Then the formaldehyde added soil was covered with polyethylene sheets and kept for 2 days.After two days the soil was uncovered to release the gas of formalin by spreading the soil mixture.The prepared soil was poured into perforated plastic pot (22 cm height and 18 cm diameter) at the rate of 3 kg soil per pot.Tomato seedlings of 11-12 cm height (28 days old) were planted in each pot previously filled with sterilized soil mixture.Different intercultural operations were done and the crop was monitored regularly.
In net-house pot culture experiment, four different treatments were used as follows:

Statistical analysis:
The experiment was done following Completely Randomized Design (CRD).The data were subjected to analysis of variance (ANOVA) and mean comparison was conducted using the least significant difference (LSD) test at 5% level of probability.Pearson's correlation coefficients were calculated on the plant parameters and nematode indexes.Differences between means were compared.

Effect of Paecilomyces lilacinus on plant height and number of leaves per plant in tomato
Tomato plants inoculated with egg masses of Meloidogyne spp.(T 1 ) highly reduced the Plant height (62.67 cm) than Control treatment (76.5 cm) as shown in Table 1.Highest plant height was recorded by P. lilacinus (78.16 cm) application followed by simultaneous application of egg masses of Meloidogyne spp.and P. lilacinus (77.00 cm).Significant difference of plant height was not obtained in the treatments T 2 (P.lilacinus), T 3 (Application of egg masses of Meloidogyne spp.and P. lilacinus, simultaneously) and T 4 (Control).Percent decrease of plant height over control was 18.08% when inoculated with egg masses of Meloidogyne spp.(Table 1).

Effect of P. lilacinus and other treatments on dry weight of root per plant and root length per plant in tomato
Comparative efficacy of P. lilacinus and other treatments on dry weight of root per plant and root length per plant in tomato was recorded (Table 3).Highest amount of dry weight of root per plant (1.04 g) was found in treatment T 2 followed by T 3 (0.93 g).Application of P. lilacinus increased amount of dry weight of root per plant over control was 85.71%, and application of egg masses of Meloidogyne spp.and P. lilacinus simultaneously increased amount of dry weight of root per plant over control was 66.07%.On the other hand, Inoculation of egg masses of Meloidogyne spp.(T 1 ) highly decreased Number of flowers per plant over control (23.21%).
In case of root length per plant, highest root length per plant (7.00 cm) was recorded in T 2 followed by T 3 (6.00).Application of P. lilacinus increased root length per plant over control by 55.55%, and application of egg masses of Meloidogyne spp.and P. lilacinus simultaneously increased root length per plant over control by 33.33%.On the other hand, inoculation of egg masses of Meloidogyne spp.(T 1 ) highly decreased number of flowers per plant over control (11.11%).

Discussion
The results of the present experiment indicated that application of P. lilacinus suppressed Meloidogyne spp.and increased plant growth parameters.In the present study, it was observed that tomato plants treated with P. lilacinus showed significant variation in plant growth parameters in comparison to control.A significant increase in plant shoots and root length, number of flowers and effective flowers, dry weight of roots was observed (Table 1~3).Improved plant growth characters by application of P. lilacinus in controlling root knot nematodes were also reported earlier by Khan and Goswami (2000).They tested the effects of P. lilacinus on Meloidogyne spp. of tomato and observed that highest root length and shoot length, fresh weight and dry weight of root and shoot were achieved when plants were inoculated with P. lilacinus to control root knot nematode.Banana plants treated with P. lilacinus significantly increase plant height, pseudostem girth, number of leaves, leaf area, shoot weight, root length and weight (Jonathan and Rajendran, 2000).
It was observed that Meloidogyne spp.retarded the growth and reduced the fresh and dry weight of the tomato plants.Apparently P. lilacinus showed effectiveness in suppressing Meloidogyne spp.. Khan et al. (2012) recorded an enhancement in growth and yield of eggplant with bio-control agents Pochonia chlamydosporia, Paecilomyces lilacinus, and Trichoderma harzianum by the suppression of galls formation.Jonathan and Rajendran (2000) reported that in banana significant reduction was observed in root gall index, egg masses, eggs per egg mass, females and soil population of the nematode treated with P. lilacinus inoculated neem cake.P lilacinus treated root samples showed many empty eggshells and an abundance of hypae were present endogenously in the eggs.Similar observations were reported on potato and betel vine due to P. lilacinus against M. incognita (Jonathan et. al., 1995).
Although the isolates of P. lilacinus show some potential as a bio-control agent, the study must be continued to increase its efficacy.Monjil and Ahmed (2017) found the fungus attacked eggs of Meloidogyne spp.and inhibit the nematode hatching from the egg masses.P. lilacinus caused substantial egg deformation in M. incognita, these deformed eggs never matured or hatched (Jatala et al., 1985).The serine protease produced by P. lilacinus might play a role in penetration of the fungus through the egg shell of the nematode (Bonants et al., 1995).Therefore, application of P. lilacinus before the nematode attack would offer greater protection to plants without damaging plant roots.P. lilacinus attacked and destroyed egg masses and juveniles of Meloidogyne spp.resulting improved plant growth.
T 1 : Inoculation of egg masses (10 egg-masses/plant) of Nematodes (Meloidogyne spp.) T 2 : Application of Paecilomyces lilacinus (10×10 5 spores/g dry weight of sample) T 3 : Application of egg masses of Nematodes (Meloidogyne and Paecilomyces lilacinus simultaneously.T 4 : Control (non-treated)Parameters of data collectionData on different parameters were collected viz., plant height, number of leaves, number of flowers, number of effective flowers (flowers converted to fruits), length of roots, number of gall and dry weight of roots.Plant height and umber of leaves per seedlings were measured after 15, 30, 45 and 60 days of planting.Number of flowers and Number of effective flowers were counted after 30, 45 and 60 days of planting.Flower which produced fruit was considered as effective flower.Two months after potting of seedling, plants were harvested and the length of the roots (cm) was measured.Number of galls in root was counted in order to compare the efficacy of P. lilacinus against Meloidogyne spp.Dry weight of roots was measured.

Table 1 . Effect of Paecilomyces lilacinus on Plant height and Number of leaves per plant
Effect of P. lilacinus on number of flowers per plant and number of effective flowers per plant in tomatoSignificantly different effect was observed among the treatments (Table2).Highest number of flowers per plant (25.26 ) was found in T 2 followed by T 3 (20.32).