In vitro Studies on the Nematophagous fungus Clonostachys rosea (TNAU CR 01) against Meloidogyne incognita, a Root-knot Nematode

Author: Shaliha B.*, G. Jothi, N. Swarnakumari and L. Rajendran

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Abstract

Meloidogyne incognita larval mortality and egg hatching were investigated using Clonostachys rosea culture filtrate. Higher concentrations of the fungal culture filtrate infested M. incognita juveniles and impaired egg hatching. The hatching rate of M. incognita eggs decreased as concentrations increased. Due to the lytic effect the morphological alteration of embryo and eggshell with hyphal penetration and internal egg colonization were observed. When compared to control, culture filtrate of C. rosea was found effective against the nematode M. incognita in terms of J2 mortality (100 percent), egg hatching inhibition (94.12 percent), and parasitization of M. incognita eggs (76.94 percent) after 96 hours of exposure time, indicating high biocontrol capability against the root knot nematode, M. incognita. Similarly different dilutions of culture filtrate significantly inhibited hatching of M. incognita eggs and larval emergence was inversely proportional to filtrate concentrations.

Keywords

Clonostachys rosea, culture filtrate, in vitro; Meloidogyne incognita, parasitism, bio-control agent, nematophagous fungus

Conclusion

To summarise, the current study found that Clonostachys rosea efficiently decreased egg hatchability and mortality of infective juveniles of root knot nematode and parasitized M. incognita eggs under in vitro studies.

References

INTRODUCTION Nematodes belong to the phylum Nematoda (Kingdom Animalia), which has over 25,000 species with a wide range of feeding habits, including animal-parasitic, plant-parasitic, bacterivorous, fungivorous, omnivorous, and predatory (Blaxter, 2003). Plant-parasitic nematodes are a major global hazard in field crops, vegetables, and fruit crops (Singh and Kumar, 2015). When compared to other plant parasitic nematodes that are polyphagous, endoparasites, and sedentary vascular root feeders, root knot nematodes (Meloidogyne spp.) are considered cryptic adversaries (Sharon et al., 2001). Meloidogyne arenaria, M. incognita, M. hapla, and M. javanica, four common species of this genus have been identified as dangerous (Dong et al., 2014). M. incognita is the most dangerous of these species due to its wide host range, rapid reproduction rate, potential to cause complex diseases in collaboration with other pathogens, and quick generation time (Vos et al., 2013). Pandey and Nayak (2018) discovered that M. incognita alters host metabolic processes, causing alterations in the infected host at the cellular, physiological, and biochemical levels which are difficult to manage. Nematicides based chemicals have been used to control plant-parasitic nematodes, which is now restricted due to environmental concerns, nematode resistance development (Abad et al., 2008), high costs, toxicity to plants, livestock, and biodiversity (Beketov et al., 2013). The need for more ecofriendly alternatives, such as the use of microorganisms such as fungi, bacteria, protozoa, viruses, nematodes, and mites and their derivatives from various sources, is urgent because most synthetic nematicides will likely be withdrawn from the market soon (Sarrocco & Vannaci 2018). In the soil, nematophagous fungi are commonly associated with nematodes. These fungi either actively parasitize nematodes with sticky branches, networks, knobs, constricting and non-constricting rings, or indirectly assault them by releasing harmful chemicals and enzymes (Zouhar et al., 2013). Clonostachys is a mycoparasite and opponent of pathogenic fungi, insects, and parasitic nematodes that is well known (Goh et al., 2020). Clonostachys rosea (syn. Gliocladium roseum) fungi are microscopic fungus that has been used as a biological agent to control plant parasitic nematodes (Trainer et al., 2014). C. rosea has showed potential lethal effects against M. incognita under in vitro condition (Wang et al., 2011). In this study, in vitro investigations such as egg hatching inhibition, juvenile mortality, egg parasitization, and parasitization in bittergourd against root knot nematode have been assessed to study the biocontrol potential of the nematophagous fungus C. rosea. MATERIALS AND METHODS A. Pure culture of root knot nematode Root knot nematodes were grown and maintained as pure culture in tomato plants under glasshouse conditions at the Department of Nematology, TNAU, Coimbatore. After confirming the species of root knot nematodes, egg masses were obtained from diseased roots, placed in a beaker containing sterile distilled water and allowed to develop for 4 days at room temperature. The clay pots were filled with a sterile pot mixture at the ratio of red soil, sand and FYM as 2:1:1. Root knot nematode juveniles were introduced at the root area of 25-day-old tomato seedlings. The pure culture egg masses were collected for future studies. Morphological Identification of M. incognita through the Posterior Cuticular Pattern Posterior Cuticular Pattern (PCP) was used to identify the species of root knot nematode. The root portions were stained with acid-fuchsin lactophenol, which was followed by a 24-hour period of plain lactophenol to remove the stain from the root tissue. The stained female posterior cuticular pattern, which includes a high dorsal arch of smooth to wavy striae but no obvious lateral incisures, identified it as M. incognita (Eisenback et al., 1981). Pure culture of fungus Clonostachys rosea (TNAU CR 01) was isolated from Ooty and provided by the Department of Nematology, TNAU, Coimbatore. Potato Dextrose Agar (PDA) medium was used to develop and maintain the fungus. Fungalculture filtrate preparation C. rosea culture filtrate was produced by culturing the fungus in potato dextrose broth. The PDB medium (100 mL in 250 mL flasks) was autoclaved for 30 minutes at 15 psi. Each flask was then infected with four (5mm diameter) scoops of the fungus from an actively developing culture on Potato Dextrose Agar under sterile conditions and incubated at 25°C for 10 days. The cultures were passed through Whatmann filter paper No. 1 at the conclusion of the incubation period to remove the mycelial mats. The resulting filtrate was assigned a concentration of 100 percent. By adding the required amount of sterilized distilled water, the concentration was prepared as 25, 50, 75, and 100 percent. Incubation test. Three ml of culture filtrate from each concentration was pipetted to 4.5cm diameter petri plates to study the effect of different concentrations of C. rosea culture filtrate on egg hatching of M. incognita. Three egg masses of M. incognita of uniform size were hand-picked and placed in each dish. As a control, egg masses were put in distilled water. Each treatment was carried out four times. The temperature of each petri dish was maintained at 25 °C. After 24, 48, 72, and 96 hours, data on egg hatching was collected. To compare the impact of culture filtrate with that of the medium, the same method was done using potato dextrose broth. Egg hatching inhibition (%) =(Total number of unhatched eggs)/(Total number of eggs) × 100 Test for mortality. Three ml of culture filtrate from each concentration was placed into each petri dish, and roughly 100 freshly hatched second stage juveniles of M. incognita in 0.2 ml distilled water were transferred to each petri dish to evaluate the effect of varying concentrations of the culture filtrate on juvenile mortality. As a control, juveniles were placed in distilled water. Each treatment was replicated four times. To compare the effect of culture filtrate and plain medium, the number of dead (unmoved) larvae in each petri dish was counted after 24, 48, 72, and 96 hours, and their percentage were calculated. Mortality (%) = (Number of dead juveniles in treatment )/(Number of juveniles inoculated) × 100 Parasitization of eggs. Each egg mass was placed in a petri plate with fungal culture before being incubated at 25±2°C. At 24, 48, 72, and 96 hours, observations were made. There were four replicates and the control eggmass in distilled water. Counting parasitized and non-parasitized eggs under a microscope were done to determine the % egg parasitism. Eggs that were infected by direct hyphal penetration or disintegration of their contents were considered as infected, but eggs that contained living juveniles or eggs from which juveniles had hatched were counted as viable (Khan, Williams, and Nevalainen 2006). Egg parasitism (%) =(Total parasitized eggs)/(Total number of eggs) × 100 Penetration study. The study was to observe the effect of C. rosea on root knot nematode penetration in bittergourd. Fungal culture filtrate (5.4×107 spores/ml) was used to inoculate fifteen days old bittergourd seedlings. 100 second-stage juveniles were injected after 7 days. Plants without fungus served as a control. The observations were taken every 24 hours. Uprooted roots were dyed with acid fuchsin – lactophenol and then destaining was done with ordinary lactophenol. Data Analysis. The AGRES programme was used to analyze the data from the above-mentioned studies, and Duncan's Multiple Range Test was used to interpret the significant means (Fisher, 1935). p=0.01 was considered as the level of significance. When the "F" test was determined to be significant, critical difference (CD) values were generated for p=0.01. RESULTS AND DISCUSSION The analysis of variance revealed a significant interaction between filtrates and concentrations (F × C), filtrates and time interval (F × T), concentrations and time (C × T), and among filtrates, their concentrations, and time (F × C × T). Effect of culture filtrates on Meloidogyne incognita egg hatching. The results revealed that there were substantial variations (p=0.01) in the concentrations of different culture filtrates. In addition, the hatching rates of M. incognita eggs were inversely linked to the concentrations of filtrates, indicating that the hatching rate reduced as the concentrations increased. After four days, control eggs had the highest hatch rate (96.54%) while eggs treated with 100 percent concentration of C. rosea culture filtrate had the lowest hatch rate (5.87%) (Table 1). The hatching of M. incognita eggs was considerably suppressed by various dilutions of the culture filtrate. Percentage of hatching was inhibited the most at 100 percent (94.12) and 75 percent (87.37) concentrations of culture filtrate, followed by 50 percent (85.5). According to Singh and Mathur (2010), eggs that contained juveniles and seemed normal as well as eggs from which juveniles had hatched out were counted as healthy. Eggs that were contaminated by direct hyphal penetration or by the dissolution of their contents were counted as infested. Egg hatching of M. incognita in varied concentrations of the medium filtrate was statistically equal in distilled water, it was inferred that the medium lacked ovistatic or ovicidal properties. Pochonia chlamydosporia showed highest egg parasitism, egg hatch inhibition and juvenile mortality of M. incognita at 25, 50, 75 and 100 percent concentrations (Annapurna et al., 2018). Effect of culture filtrate on Meloidogyne incognita juvenile mortality. The concentration of fungal filtrates and the length of exposure were shown to be directly linked to juvenile mortality. At 100 percent concentration of C. rosea, greatest death rate of J2 (63.75 percent) occurred after 24 hours of exposure. The mortality of juveniles increased as the concentrations of the filtrates progressively increased. After 96 hours of exposure at 100% concentration of C. rosea filtrate, the maximum mortality (100%) occurred. In C. rosea filtrate at 25% concentration, the lowest mortality was 62.75 percent (Table 2). The findings also revealed that M. incognita second-stage juveniles were more susceptible to these fungus secondary metabolites. The percentage of highest levels of juvenile mortality were seen in the culture filtrate concentrations of 100 percent (100.00) and 75 percent (86.25), followed by 50 percent (76.75). Nematodes that were rigid and elongated with head and tail sometimes slightly bent were considered as immobilized and if they did not react when probed with a fine needle were considered as paralyzed The most promising fungus for controlling M. incognita was C. rosea, and its nematicidal impact was proven (Wang et al., 2011). This fungus may generate a huge number of conidia in a short period of time, which adhere to nematodes and germinate in the body cavity. Proteases, collagenase, and chitinase are hydrolytic enzymes produced by the fungus, and they may be implicated in nematode cuticle penetration and host cell disintegration. Hussain (2017) found that incubation in C. rosea resulted in 70.2 percent inactivation of M. hapla J2 after 48 hours and an 86 percent inactivation after 72 hours. M. incognita egg parasitization. In the study of egg parasitization, C. rosea had the highest rate (63.97%) (Table 3). The egg shells of the fungi-infested eggs disintegrated and shrank abnormally. Immature eggs that had been parasitized by fungus had destroyed embryos, protruding interior contents, and spores filled the eggs. The eggs, either infected by direct hyphal penetration or disintegration of their contents, were counted as parasitized (Khan, Williams, and Nevalainen 2006), while eggs that contained live juveniles and eggs from which juveniles had hatched were counted as unparasitized. Due to lytic effect the morphological alteration of embryo and eggshell, with hyphal penetration and internal colonization were observed. Filtrates from cultures of Fusarium spp., Paecilomyces lilacinus, and Pochonia chlamydosporia were toxic to M. incognita second stage juveniles, inhibited hatching and/or suppressed egg or J2 populations on plants (Meyer et al., 2004). C. rosea effect on juvenile penetration. Under glass house conditions, second stage juvenile penetration on bittergourd roots were observed up to 7 days after inoculation (DAI) on 3, 4, 5, and 7 DAI. The number of nematode penetration in C. rosea treated seedlings was reduced considerably by 78.82 percent, 80.43 percent, 81.20 percent, 75.56 percent and 76.94 percent respectively, compared to control (Table 4). Regardless of DAI, C. rosea prevented juvenile penetration in roots, according to the outcomes. C. rosea showed the greatest decrease (76.94 percent) after 7 days when compared to the control. In order to promote colonization and provide resistance against harmful interactions, fungal partners release bioactive molecules such enzymes, short peptide effectors, and secondary metabolites (Zeilinger et al., 2015). Antibiosis, direct parasitism, and enzyme secretion are the techniques used by C. rosea to kill nematodes (Iqbal et al., 2018). Its hyphae can parasitize M. incognita eggs and adults. Conidia adhere to the nematode's cuticle at first, then germinate and pierce the nematode's cuticle, killing it (Zhang et al., 2008). The production of nematotoxic secondary metabolites has also been described, however the precise site of action has yet to be determined (Song et al., 2016). Approximately 38 metabolites from C. rosea have been described so far, with a considerable structural variation (Abdel-Wahab et al., 2019; Supratman et al., 2019). Gliocladin C and 5-n-heneicosyl resorcinol displayed nematotoxic action against zooparasitic nematodes (Song et al., 2016). C. rosea mode of action against nematodes, according to Iqbal et al. (2018), is antibiosis rather than parasitism. Chitinase and proteases are enzymes that hydrolyze the nematode cell wall’s-(1-4) glycosidic linkages (Yang et al., 2010). Nematicidal action of culture filtrates of V. chlamydosporium against M. javanica may be attributed to the production of certain enzymes (Segers et al., 1994) and toxins like Verticillin A, B and C which help in weakening and dissolving the barriers of its hosts. Species of Aspergillus, Penicillium, Talaromyces, Curvularia and Aternaria are known to produce toxins and antibiotics like aflatoxin, penicillin, vermiculin, vermicillin, talaron, vermistatin, viridin, fusaricacid, rhizopin, lilacinin, leucinostatin, P-168 and phytoalternarin. The findings showed that the Clonostachys rosea fungus parasitizes nematodes by excreting chemicals that can paralyze or kill J2 of the root-knot nematode, M. incognita. A number of nematophagous fungi are known to have proteolytic and chitinolytic activities which cause alteration in eggs’ cuticular structure, changes in egg shell permeability or cause perforations in the cuticle which allows seepage of toxic metabolites into the eggs and cause physiological disorders (Tariq Mukhtar and Ijaz Pervaz 2003).

How to cite this article

Shaliha B., G. Jothi, N. Swarnakumari and L. Rajendran (2022). In vitro Studies on the Nematophagous fungus Clonostachys rosea (TNAU CR 01) against Meloidogyne incognita, a Root-knot Nematode. Biological Forum – An International Journal, 14(2a): 398-408.