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Nematicidal activity of Paecilomyces lilacinus 6029 cultured on Karanja cake medium Q3

Abhishek Sharma a, Satyawati Sharma a, *, Manu Dalela b a b

Centre for Rural Development & Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Centre for Biomedical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 May 2014 Received in revised form 19 August 2014 Accepted 22 August 2014 Available online xxx

Antagonistic fungi parasitize root-knot nematodes through secretion of extracellular hydrolytic enzymes and secondary metabolites. In present study, in vitro bioassay showed that Paecilomyces lilacinus 6029 culture filtrate from Karanja cake medium killed 100% Meloidogyne incognita larvae while only 78.28% mortality was recorded by Czapeck-Dox filtrate within 12 h of exposure. The filtrate, irrespective of culture medium, was found to be more nematotoxic when incubated for 15 days. Fourier Transform infrared spectroscopy predicted the presence of phenolic and alcoholic compounds in the filtrate. Furthermore, the active metabolites in fungal filtrate were partially characterized. pH stability test revealed that nematotoxicity of the filtrate appeared at all range of pH with low pH filtrate possessing more toxicity against M. incognita. Interestingly, buffers of same pH value did not show any nematicidal effect. No significant difference in nematicidal activity was observed between boiled (98.2% mortality) and unboiled culture filtrate (100% mortality). Ethyl acetate and lyophilized aqueous extracts produced higher nematicidal activity than a hexane extract indicating polar nature of active compounds produced by P. lilacinus 6029. © 2014 Published by Elsevier Ltd.

Keywords: Paecilomyces lilacinus Meloidogyne incognita Culture filtrate Karanja cake Nematicidal

1. Introduction Root-knot nematodes (Meloidogyne spp.) are the most frequently observed and damaging plant-parasitic nematodes in crops [1]. They can impair root function and act as nutrient sinks thus reducing crop yield [2,3]. Control of plant-parasitic nematodes in crop production systems currently relies on chemical nematicides. However, apprehensions related to environmental and health hazards necessitate the urge for new and alternative biological means of nematode control. Biological control of nematodes concerns microbial agents such as bacteria and fungi that are nematophagous or antagonistic to nematodes [4]. Fungi are perhaps the most important parasites and predators of nematodes [5]. Li et al. [6] reviewed 179 nematicidal fungal metabolites isolated from a variety of deuteromycete, ascomycete and basidiomycete fungi. Anke and Sterner [7] reviewed nematicidal metabolites from higher fungi. A new peptide called omphalotin was obtained from

* Corresponding author. Tel.: þ91 11 2659 1116; fax: þ91 11 2659 1121. E-mail addresses: [email protected], [email protected] (S. Sharma).

the fungus Omphalotus olearius and this peptide has shown to have a nematicidal activity similar to the commercial nematicide ivermectin [8]. Antagonistic fungi produce chemically diverse compounds (metabolites) causing severe adverse effects on crucial biological processes of Meloidogyne spp. [9]. Many workers have studied the toxic effect of culture filtrates of different fungi on Meloidogyne larvae [10e12]. Since bioactive metabolites have potential application as novel nematicides, most of the research are focused on identification and isolation of such compounds [13]. Concurrently, characterizations of these nematicidal metabolites are of paramount importance before developing and applying them as bionematicide in various agro-climatic conditions. Paecilomyces lilacinus, a very promising nematophagous fungus, is also known to secrete certain secondary metabolites and enzymes [14,15]. In our previous study, we developed Karanja cake based medium conditions for obtaining high yields of P. lilacinus 6029 [16]. The following study has been undertaken to investigate a) the role of Karanja cake medium and incubation period on nematicidal efficacy against Meloidogyne incognita and b) partial characterization of active nematicidal metabolites of strain 6029 of P. lilacinus cultured on Karanja cake based medium.

http://dx.doi.org/10.1016/j.micpath.2014.08.007 0882-4010/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Sharma A, et al., Nematicidal activity of Paecilomyces lilacinus 6029 cultured on Karanja cake medium, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.08.007

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subsequently transferred to sterile distilled for an hour to ascertain their mortality. If they failed to regain their mobility and appeared straight, they were considered dead. Each treatment was maintained in a triplicate.

2. Materials and methods 2.1. Microorganism and culture filtrate preparation Paecilomyces lilacinus 6029 was procured from Indian Type Culture Collection (ITCC), Indian Agricultural Research Institute, PUSA, New Delhi. The spores of the fungal strain were harvested from the slant surface by pouring sterile water (10 ml) emulsified with 0.1% tween-80 and subsequent stirring at 120 rpm. This spore suspension was used to inoculate subsequent flasks (100 ml) containing Czapeck-Dox broth (CDB) and Karanja cake based broth having a C/N ratio of 35.88 and pH 5.9 [16]. The fungal culture was incubated for 7 and 15 days in the dark at 27  C. To obtain cell free culture filtrate, fungal biomass was filtered through Whatman No.1 filter paper.

2.4. Fourier transform infrared (FTIR) spectroscopy Infrared spectrum of metabolites present in culture filtrate was studied using ATR-FTIR (Attenuated total reflectance-Fourier transform infrared) Spectrophotometer PerkineElmer 1600 to study the functional groups on the surface of the culture filtrate between the spectral range of 4000 and 400 cm1 at a scan speed of 16 cm/s. Washed and dried samples was kept on the diamond probe of the ATR for the spectroscopic analysis. 2.5. Statistical analysis

2.2. Partial characterization of active metabolites To determine the effect of pH on nematicidal efficacy, culture filtrate was adjusted to pH 3.5, 5, 7 and 9 and then filtrate of varied pH were subjected to bioassay. For thermo stability test, culture filtrate was boiled for 5 min and subsequently tested for nematicidal efficacy. To determine the solubility of nematicidal metabolites, the culture filtrate (5 l) was exhaustively extracted with hexane. The non polar hexane fraction was subsequently concentrated to a thick yellow liquid (0.65 g). The aqueous portion was further extracted using ethyl acetate as the solvent. The ethyl acetate fraction was again concentrated using a rotary evaporator to give 0.83 g of colorless liquid. The remaining aqueous fraction was concentrated through lyophilization. Each fraction was suitably diluted with emulsified water (containing 0.5% Tween-80) to afford final concentrations of 0.1, 1, 5, 10, 50 and 75 mg/ml. 2.3. Nematicidal activity assays The pure population of M. incognita was maintained on brinjal (Solanum melongena) roots growing in earthen pots with autoclaved soil at micromodel complex, IIT Delhi. Juveniles of M. incognita were collected after three days of incubation of egg masses in sterile distilled water. An in vitro bioassay was carried out in 24 well plates. In each well, 1 ml of test sample and 0.5 ml of water containing approximately 150e200 freshly hatched juveniles of M. incognita were added. The numbers of immobilized juveniles were counted after regular intervals of 3, 6, 12 and 24 h using a stereoscopic microscope. Apparently, immobilized juveniles were

Duncan's multiple range test (P < 0.05) level was employed to test for significant differences between treatments using SPSS software. LC50 were determined through probit analysis method described by Finney (1971) using Stat Plus 2009 software. 3. Results and discussion 3.1. Effect of Karanja cake medium and incubation period Filtrate of P. lilacinus 6029 obtained from our optimized Karanja cake medium (FKB) and Czapeck-Dox broth (FCDB) showed significant (P < 0.05) nematicidal effects killing the second-stage of juveniles of M. incognita to a varying extent (Table 1). On comparing the nematicidal efficacy of FKB, FCDB and controls (Karanja broth alone and CDB alone) within 3 h of exposure, it was observed that no mortality was achieved by Karanja broth alone but FKB exhibited significant toxic effect on the juveniles killing 62.18% of them while FCDB killed only 43.21% nematodes. This reveals that the Karanja cake as nutrient source increased the nematicidal activity of P. lilacinus filtrate. CDB medium did not play any effective role in killing nematodes as only 6.3% nematodes were found dead after 24 h in 15 days of incubated medium. According to Burow et al. [17], some plant unsaturated fatty acids have been shown to influence mycotoxin production in Aspergillus niger. Since Karanja cake also possesses unsaturated fatty acids, it is interesting to speculate that it could have triggered the production of certain secondary metabolites in the filtrate rendering more nematotoxicity than the filtrate from the same fungus in CDB. Some workers claimed that nutrient and other culture conditions could cause a shift in the

Table 1 Effect of Karanja cake medium and incubation period on the culture filtrate of P. lilacinus 6029 against M. incognita larvae. Treatments

Incubation period

Mortality (%) 3h

6h

12 h

24 h

FKB

0 day 7 days 15 days 0 day 7 days 15 days 0 day 7 days 15 days 0 day 7 days 15 days

0.0c 41.25 62.18 0.0a 10.35 43.21 0.0c 0.0c 0.0c 0.0a 0.0b 0.0c

6.71 ± 1.06b 66.23 ± 1.83c 90.12 ± 1.53b 0.0a 32.74 ± 2.13c 56.24 ± 1.28c 6.68 ± 1.03b 7.12 ± 1.12b 11.26 ± 1.01b 0.0a 0.0b 3.1 ± 0.52b

10.18 ± 1.0a 73.45 ± 2.07b 100.00a 0.0a 49.17 ± 1.10b 78.28 ± 1.34b 10.12 ± 0.96a 12.56 ± 1.80a 16.8 ± 1.23a 0.0a 1.8 ± .18a 5.8 ± 0.76a

11.12 ± 1.48a 80.92 ± 1.45a 100.00a 0.0a 67.65 ± 0.9a 86.35 ± 1.89a 11.04 ± 1.59a 13.86 ± 1.57a 17.6 ± 1.19a 0.0a 1.9 ± 0.16a 6.3 ± 0.62a

FCDB

KB alone (Control 1)

CDB alone (Control 2)

± 1.21d ± 2.09c ± 2.09d ± 1.64d

In each row, data followed by the same letter are not significantly different at P < 0.05 by Duncan's multiple range test. KB: Karanja cake broth; FKB: Culture filtrate from Karanja cake broth; CDB: Czapeck's Dox broth; FCDB: Culture filtrate from Czapeck-Dox broth.

Please cite this article in press as: Sharma A, et al., Nematicidal activity of Paecilomyces lilacinus 6029 cultured on Karanja cake medium, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.08.007

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Fig. 1. FTIR spectra of fungal culture filtrate of P. lilacinus 6029 obtained from Karanja cake medium incubated for 15 days (FKB 15) and 7 days (FKB 7) respectively.

biogenic pathways in microorganism leading to production of bioactive compounds not known to be associated with them [18,13]. Apart from nutrient medium, incubation period also played a significant role in enhancing the nematicidal efficacy of the P. lilacinus 6029 filtrate. The fungus, irrespective of type of media, produced less nematicidal filtrate when incubated for one week. Data revealed maximum mortality of 80.92% and 67.65% within 24 h of treatment by FKB and FCDB respectively when incubated for 7 days. However, the nematicidal activity enhanced with increase in incubation period. 100% mortality was recorded for FKB at 12 h when incubated for 15 days. This might be due to accumulation as well as secretion of secondary metabolites late in fungal growth as predicted by FTIR study (Fig 1). The comparative FTIR spectrum of FKB procured after 15 days (FKB 15) and 7 days (FKB 7) of incubation shows the presence of stretching vibration of the group CeO at 1102 cm1 and CeH at 2982 cm1 only in FKB 15 suggesting that some phenolic and alcoholic compounds might be secreted with time in culture broth. It has long been known that filamentous fungi viz. P. lilacinus, A. niger etc. generally secrete secondary metabolites during the stationary phase i.e. at the commencement of sporulation [19]. The inference of present study is also in conformity with the study conducted by Mahajan et al. [20] who reported the involvement of phenolic compounds as nematicidal agents. Gapasin et al. [21] revealed the presence of five phenolic compounds in active fraction of P. lilacinus filtrate when incubated for 60 days.

3.2. Partial characterization of nematicidal metabolites The data pertaining to pH stability test is shown in Table 2. The nematicidal efficacy of FKB at pH 3.5 and 5 was statistically same, killing 100 and 98.21% of Meloidogyne incognita larvae. When pH of the filtrate was raised up to 9, the nematicidal activity significantly declined. At 3 h of exposure, no mortality was observed while at 24 h only 59.67 and 56.34% mortality was observed by the filtrate having pH value 7 and 9 respectively. Experiments with different buffers (acidic, neutral and alkaline) showed that pH as such was not directly responsible for the nematicidal activity of acidic metabolites as any buffer at all given exposure periods showed no mortality. P. lilacinus is known to secrete acetic acid [22] and some other short-chain fatty acids [13]. However, much of the real interesting finding of this experiment was the effectiveness of these acids at low pH only. The possible rationale for this finding could be attributed to low pKa value of acids present in the filtrate. This means at higher pH, weak acids would get dissociated into ionic forms and hence unable to permeate freely inside nematode surface [23]. Glycoprotein present on nematode's surface produces hydrophilic forces thereby adsorbing ionic substances on the surface itself and preventing them to enter inside the nematode [24]. Hence, we propose that P. lilacinus filtrate at higher pH are relatively less toxic to nematodes than at low pH. Our work is in partial agreement to Cayrol et al. [25] who concluded that low pH is necessary for increased toxin

Table 2 Effect of pH on toxic activity of P. lilacinus 6029 filtrate against M. incognita. pH

Larval mortality (%) 3h

6h

3.5 (filtrate) 3.5 (buffer) 5 (filtrate) 5 (buffer) 7 (filtrate) 7 (buffer) 9 (filtrate) 9 (buffer) Control (water)

62.18 ± 1.09a 0.0c 60.46 ± 2.25b 0.0c 0.0c 0.0c 0.0c 0.0c 0.0c

90.12 0.0d 88.24 0.0d 30.72 0.0d 28.31 0.0d 0.0d

± 1.53a ± 2.1b ± 2.26c ± 2.01c

12 h

24 h

100 ± 0.0a 5.6 ± 1.12d 93.53 ± 1.59b 0.0e 48.91 ± 2.21c 0.0e 45.72 ± 2.19c 0.0e 0.0e

100 ± 0.0a 5.7 ± 1.31c 98.21 ± 1.55a 0.0e 59.67 ± 2.06b 0.0e 56.34 ± 1.52b 0.0e 1.2 ± 0.45d

In each column, data followed by the same letter are not significantly different at P < 0.05 by Duncan's multiple range test.

Fig. 2. Effect of boiling on toxic activity of filtrate against M. incognita.

Please cite this article in press as: Sharma A, et al., Nematicidal activity of Paecilomyces lilacinus 6029 cultured on Karanja cake medium, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.08.007

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Table 3 Effect of different solvent fractions of P. lilacinus 6029 on the mortality of second-stage juveniles of M. incognita. Dose (mg/ml)

0.1 1 5 10 50 75 Water (Control) LC50 (mg/ml)

Mortality % by Ethyl acetate fraction

Mortality % by Hexane fraction

Mortality % by lyophilized water fraction

6h

6h

6h

d

0.0 0.0d 0.0d 9.2 ± 1.08c 25.9 ± 2.11b 30.3 ± 1.45a 0.0d 217.08

12 h f

0.0 15.8 ± 1.23e 26.72 ± 1.12d 33.5 ± 1.76c 61.43 ± 1.95b 75.7 ± 1.23a 0.0f 21.69

24 h f

11.2 ± 1.39 29.5 ± 1.75e 50.8 ± 1.35d 73.9 ± 2.53c 82.1 ± 1.62b 100 ± 0.0a 0.0f 3.03

0.0 0.0 0.0 0.0 0.0 0.0 0.0 e

12 h b

0.0 0.0b 0.0b 0.0b 0.0b 7.5 ± 0.98a 0.0b e

24 h c

0.0 0.0c 0.0c 7.2 ± 1.52b 8.6 ± 1.92b 17.5 ± 1.21a 0.0c >1000

b

0.0 0.0b 0.0b 0.0b 20.2 ± 2.11a 23.5 ± 2.23a 0.0b 444.32

12 h e

0.0 0.0e 8.6 ± 1.17d 21.4 ± 1.29c 32.4 ± 1.84b 41.6 ± 2.35a 0.0e 164.62

24 h 0.0f 7.2 ± 0.67e 23.5 ± 1.44d 33.7 ± 2.06c 40.2 ± 2.43b 58.9 ± 1.29a 0.0f 57.39

In each column, data followed by the same letter are not significantly different at P < 0.05 by Duncan's multiple range test.

production but that so-produced toxin acts in a wide range of pH values. Both unboiled and boiled culture filtrate exhibited nematicidal activity at all exposure periods (Fig. 2). The efficacy of both boiled and unboiled filtrates was significantly at par (P < 0.05) causing 98.24 and 100% juvenile deaths respectively at maximum exposure of 24 h. This implies the thermo stability of active metabolites of P. lilacinus 6029. Findings of Zareen et al. [26] give further approval of thermo stability of fungal metabolites when they observed that active principles of Fusarium solani in the filtrate retained their nematicidal activity after heating. The results pertaining to the in vitro bioassay of the different solvent fractions of culture filtrate on J2 larvae of M. incognita are shown in Table 3. Data revealed that highest concentration (75 mg/ ml) of ethyl acetate fraction showed remarkable nematicidal property bestowing 100% mortality within 24 h of experimental period with LC50 value of 3.03 mg/ml. Lyophilized aqueous filtrate resulted in 58.9% mortality at 75 mg/ml concentration after 24 h. Hexane fractions showed very poor nematicidal property as mortality ranged from 0 to 23.5% with 0.1e75 mg/ml concentrations. From the data, it is inferred that the active principles in culture filtrate are polar in nature. Our result affirmed the findings of Siddiqui et al. [27] in which ethyl acetate fractions of P. lilacinus were more effective than hexane fractions in the suppression of Meloidogyne javanica larvae. Taken as a whole, our data implies that the active principles are majorly non-proteins in nature with high polarity and therefore, extreme pH and temperature did not considerably affect the nematicidal efficacy of the culture filtrate against M. incognita. 4. Conclusions In present study, maximal nematicidal activity was exhibited by filtrate of P. lilacinus 6029 incubated for 15 days in Karanja cake medium correlating for the first time Karanja cake with the pathogenicity of the fungus. Inconsistent nematicidal activity of P. lilacinus 6029 during characterization study postulates the presence of chemically diversified metabolites. Although the field conditions are very robust and unpredictable, this work could at the least, provide useful support in interpreting effectiveness of bioactive compounds of P. lilacinus under varied nutrient and environmental conditions. Nevertheless, a more sensitive and complete characterization of bioactive compound(s) of P. lilacinus through Nuclear Magnetic Resonance (NMR) spectroscopy would be of great help before their practical applications in the field. Acknowledgments The authors gratefully acknowledge financial support provided by the National Oilseed and Vegetable Oil Development (NOVOD) Board, Gurgaon, India, for carrying out the research.

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[26] Zareen A, Siddiqui IA, Aleem F, Zaki MJ, Shaukat SS. Observations on the nematicidal effect of Fusarium solani on the root-knot nematode, Meloidogyne javanica. J Plant Pathol 2001;83:207e14. [27] Siddiqui BS, Farhana A, Ghasuddin M, Faizi S, Naqvi SNH, Tariq RM. Two insecticidal tetranortriterpenoids from Azadirachta indica. Phytochemistry 2000;53:371e6.

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Nematicidal activity of Paecilomyces lilacinus 6029 cultured on Karanja cake medium.

Antagonistic fungi parasitize root-knot nematodes through secretion of extracellular hydrolytic enzymes and secondary metabolites. In present study, i...
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