Recent Acheievement-Nematology – ICAR-SUGARCANE BREEDING INSTITUTE

Recent Achievements - Nematologyy

Plant Parasitic Nematodes: Biological Control

♣ Nematode antagonistic fungi viz., Paecilomyces lilacinus, Trichoderma viride and Trichoderma harzianum were found to be effective in suppressing root-knot and lesion nematodes.

♣ Plant growth promoting rhizobacterial (PGPR) isolates with nematode suppressive ability against root-knot and lesion nematodes were identified

♣ Fungal and rhizobacterial isolates with nematode suppression and phosphate solubilization ability were identified.

♣ Low cost technologies based on locally available substrates were developed for mass production of nematode biocontrol agents.

♣ Nematophagous fungus Arthrobotrys oligospora has been isolated from sugarcane field and its efficacy was tested against sugarcane phytonematodes

♣ Mass production of nematophagous fungi Arthrobotrys oligospora was standardized in liquid molasses medium.

♣ Arbuscular mycorrhizal fungi (AMF) has been isolated from sugarcane fields, and its biocontrol efficacy was tested against lesion and root knot nematodes.

Plant Parasitic Nematodes: Integrated Control

♣ In field, bioagents Paecilomyces lilacinus Pochonia chlamydosporia and Pseudomonas flourescens in combination with INM practice showed reduction in nematode population and increase the yield of sugarcane.

♣Integrated Nematode Management in combination with biofertilizers (Azospirillum and Phosphobacteria) was evaluated in field condition against sugarcane phytonematodes.

♣Combined application of Arbuscular mycorrhizal fungi viz., Glomus mosseae and G. fasciculatum along with three nematode bioagents viz., Arthrobotrys oligospora, Paecilomyces lilacinus and Pochonia chlamydosporia on sugarcane cv. Co.86032 was evaluated against lesion nematode Pratylenchus zeae and root knot nematode Meloidogyne javanica. Synergistic effect was noticed with AMF and other bioagents in controlling the nematode population.

♣ A Bio intensive Integrated Nematode Management (INM) package based on eco-friendly components was developed involving green-manuring with legumes like sunhemp or daincha before planting; application of organic amendments such as of cured pressmud/Farm Yard Manure (FYM) @ 25 t/ha or neem/castor oil cake @ 2t/ha during field preparation or mulching with cane trash @ 5t/ha and its incorporation into soil during earthing-up operation; intercropping with short duration legumes like soybean and green gram; application of biocontrol agents like Paecilomyces lilacinus or Verticillium chlamydosporium or Trichoderma viride or Pseudomonas fluorescens @ 20 kg/ha at the time of planting and application of nematicides like carbofuran or phorate @ 3kg a.i./ha at two slits i.e. half the dose at the time of planting and the second half 90 days after planting and it should be followed up with application of organic amendments or green manuring in the next season to sustain the nematode control achieved using nematicides.

Entomopathogenic Nematodes: Isolation, morphological and molecular characterization of EPN

♣ Entomopathogenic nematodes (EPN) belong to Heterorhabditis and Steinernema spp. has been isolated from different cropping system and more than two dozen EPN isolates are being maintained in the culture collection. Morphological and molecular characterization of species / isolates of Heterorhabditis and Steinernema was attempted using RAPD-PCR, Protein markers and sequencing of rDNA ITS1, ITS2 and 5.8S rDNA.

♣ Twenty four EPN isolates were molecularly characterized by analysis of genomic DNA sequences. Primers used in the Polymerase Chain Reaction (PCR) were specific for the internal transcribed spacer (ITS) region of the nuclear ribosomal genes. Amplifications were carried out in a thermocycler. Clear bands of 700-800 bp appeared for Steinernema and Heterorhabditis. The PCR products were sequenced. Among the eleven Heterorhabditis isolates nine (Heterorhabditis isolates DSM8, DSM22, DSM67, DSM78, DSM81, DSM85, BNR, LN2 and Karnal3) having 99 to 100% similarity with H. indica and two have similarity with H. bacteriophora. Among 13 Steinernema isolates, nine (isolates SBI17, SBI18, 230, 374, citrus isolate, agali, HIII, karnal, and puli1) having 98 to 99% similarity with S. siyamkayi. Isolate LN1 having 83% similarity with S. glaseri.

♣ Genetic variability in environmental stress tolerance traits (heat, desiccation, and hypoxia) and fitness traits (virulence and reproductive potential) among isolates of entomopathogenic nematodes were studied.

Entomopathogenic Nematodes: Exploitation of entomopathogenic nematodes for sugarcane pest control

♣ Entomopathogenic nematode isolates possessing relatively higher potential and virulent against sugarcane internode borer Chilo sacchariphagus indicus, and shoot borer Chilo infuscatellus were identified. Field application technology of EPN with suitable antidesiccants materials and UV protectants were identified and standardised for foliar application of EPN in the foliar system to overcome desiccation of EPN and to improve its efficacy against foliar pests in the field.

♣ Compatibility studies of entomopathogenic nematodes with entomopathogenic fungi Beauveria bassiana and B. brongnartii were studied in sugarcane internode borer C. sacchariphagus indicus and shoot borer C. infuscatellus

♣ EPN were isolated from white grub infested sugarcane fields. Fifteen EPN exclusively isolated from white grub endemic area and were maintained in the culture collection of ICAR-SBI, Coimbatore.

♣ Synergistic effect of insecticide Imidacloprid and EPN Heterorhabditis indica (isolates DSM78 and BNR) and H. bacteriophora (Hb) noticed with 100% mortality 1st instar white grub H. serrata and 44 to 100 % mortality of 3rd instar grubs than individual inoculations of either EPN or Imidacloprid.

♣ Combined application of Imidacloprid and EPN Heterorhabditis indica (BNR isolate), H. indica (DSM78) and Steinernema siyamkayai (karnal) recorded 33.4 to 100% mortality of 3rd instar white grub H. serrata under pot culture and 60 to 75% mortality under microplot conditions.

♣ Under field experiment at Coromandel Sugars Ltd, Krishnarajpet, Karnataka the synergistic effect of insecticide Imidacloprid and EPN was evaluated on sugarcane Cv. Co86032. It was observed that, about 28.5 to 100 % mortality of grubs recorded third week after EPN inoculation. Maximum (100%) was recorded in EPN and Imidacloprid combined treatment followed by Imidacloprid alone treatment (72.7%).

♣ In another two field trials against white grub H. serrata with sugarcane cultivar Co. 86032, the per cent reduction of grub population due to EPN was 43 to 77

Entomopathogenic Nematodes:Mass production and formulation of EPN

♣ In vivo and In vitro mass production techniques have been standardized for H. indica, H. bacteriophora, Steinernema carpocapsae, S. Glaseri and S. siyamkayi

♣ Monoxenic culturing of EPN, S. glaseri and H. indica was standardised in lipid agar medium on petri plates. Among the different media tried for mass production of S. glaseri, chicken egg, palm oil and yeast extract medium recorded maximum production of S. glaseri. The efficacy of in vitro produced EPN was tested against G. mellonella. About 33 to 100 per cent mortality of G. mellonella larvae recorded with in vitro produced IJs.

♣ Formulation of EPN has been standardised on Talc powder, alginate granules and gel.

Toxins Studies

Exploration and exploitation of novel insecticidal toxins and antimicrobial compounds from symbiotic bacteria Photorhabdus / Xenorhabdus associated with entomopathogenic nematodes

♦ Insecticidal and fungicidal activity of bacterial symbiants, Photorhabdus and Xenorhabdus associated with EPN were explored which paved a new direction to exploit these symbiotic bacteria for insect pests control and pathogenic fungi management

Isolation of symbiotic bacteria, biochemical and molecular characterization

♦ Symbiotic bacteria were isolated from Galleria mellonella infested with EPN Biochemical characterization tests of all bacterial isolates was done to identify the gram negative rods. The following tests were done such as citrate utilization test, lysine utilization test, ornithine utilization, urease detection, phenylalanine deamination, nitrate reduction, hydrogen sulphyte production test and five carbohydrate utilization tests including glucose, adonitol, lactose, arabinose and sorbitol.

♦ Molecular characterization of bacterial isolates was done by amplification of the 16S rDNA gene and the 16S rDNA sequences were compared with the database from the sequence gene bank at the National Centre for Biotechnology Information (NCBI). All the Xenorhabdus isolates have maximum similarity with Xenorhabdus stockiae and Photorhabdus had similarity with P. luminescens.

Testing insecticidal activity of Xenorhabdus against white grub Holotrichia serrata and greater wax moth larvae Galleria mellonella

♦ The bacterial cell and cell free culture filtrates of five Xenorhabdus stockiae were tested its insecticidal activity against first instar white grub Holotrichia serrata and full grown larvae of Gallleria mellonella. All the bacterial isolates were known to have insecticidal properties and caused mortality of the Galleria. Among the isolates, X. stockiae (SBIXSCBE10) recorded maximum mortality (96.6%) of white grub and X. stockiae (SBIXS52) caused 83.3% mortality of G. Mellonella.

Purification of toxic metabolites of Xenorhabdus stockiae

♦ Purification of insecticidal metabolites of two symbiotic bacteria viz X. stockiae (SBIXS52) and X. stockiae (SBIXSD4) was done using cell free supernatant using ethyl acetate. The ethyl acetate fraction was bioassayed for its insecticidal activity against Galleria mellonella