Biological Control Agents

Question 1

Agronomic insect pests in Agriculture

Answer 1

• Agriculture is vital for food security and country GDP.
• Problematic insects cause a decline in agricultural yield every year.
• Some problematic insects show signs of drug resistance.
• An eminent solution to this problem is required.

Question 2

Chemical pesticides

Answer 2

• Chemical pesticides they are not specific to their insect range and therefore pose
  a threat into humans and any herbivore species feeding in the crops,
• Both the use of chemical pesticides and fertilizer initiate oligodynamic process in
  the soil.
• Chemical fertilizers abrogate the fertilizer cycle in the soil and kill beneficial
  bacteria in a soil.
• Both Chemical pesticides and fertilizers changes physiochemical properties of the
  soil making it inhabitable for soil native organisms.

Question 3

oligodynamic

Answer 3

Oligodynamic is a process where by there is a deposit of metals (such as Cu, Zn, Cr) leading to change in soil pH

Question 4

Importance of EPNs in agriculture

Answer 4

• EPNs are specific to their insect targets unlike to chemical integrated
  pesticides.
• EPNs provide a nutritional circle and important for nitrogen fixation in soil
  which is very beneficial to the crops.
• EPNs do not alter any soil pH and do not possess any health threats to
  human and any other herbivores species grazing in the field unlike the
  chemical fertilizers.

Question 5

Biological control

Answer 5

using natural enemies to control problem pests

Question 6

(IPM) system

Answer 6

integrated pest
management (IPM) system
* They infect and kill various insect pests that are soil-dwelling and are safe
to non-target organisms hence they are largely used as biological control
agents

Question 7

Molecular identification of EPNs

Answer 7

■ Molecular approaches include polymerase chain reaction to amplify highly
conserved regions in the DNA, DNA sequencing techniques and phylogenetic
analysis
■ EPNs studies make use of sequence data from whole genomes, nuclear and
mitochondrial genetic loci to understand phylogenetic relationships
○ Nuclear genes include external non-transcribed spacer (NTS), 18S (small
subunit), 28S (large subunit) and the internal transcribed spaces (ITS)
which is the non-functional DNA between the 18S and 28S ribosomal DNA
(rDNA) genes

Question 8

Phylogenetic analysis

Answer 8

18S rRNA sequence aligned using ClustalW function on MegaX
§Maximum likelihood trees constructed using the phylogenetic function and
Kimura 2-parameter model, with bootstrapping of 1000 replicates
§Gene sequences of the reference strains are obtained from Genbank

Question 9

Morphological identification

Answer 9

■ Morphological identification is difficult because nematodes have
morphological characteristics that are relatively conservative
■ The use of morphological and molecular approaches has been proven
sufficient
○ has an important role to play in improving the EPN taxonomy, which has
further contributed to a greater understanding of the EPNs biodiversity,
geographic distributions, ecology, behaviours, geographic distributions and
co-evolution

Question 10

Identification of EPNs

Answer 10

G. mellonella larvae
Colour change may be used to predict the EPNs

Question 11

Soil sampling

Answer 11

■ Soil samples collection
■ Collected from a depth of 20 cm with sterilized hand shovel
■ Placed in clean plastic containers
■ Soil kept moistened to prevent desiccation

Question 12

Trapping of
nematodes

Answer 12

■ Galleria baiting method
■ Larvae will be placed in separate plastic containers an stored at 25 ̊C in the dark
■ Observed daily and symptoms recorded
■ Collected after 48- 72 hours and left to dry at room temperature

Question 13

Light microscopy

Answer 13

■ 30 first generation, 30 second generation and 20 IJs
■ Killed with 80% triethanolamine-formalin (TAF) for 24 hours
■ Fixed with Seinhorst I solution (20 parts of 95% ethanol, 1 part glycerine and 79 parts of water)
■ Placed in 35 ̊C desiccator and filled with Seinhorst II solution (95 parts of 95% ethanol and 5
parts of glycerine)
■ Placed in 40 ̊C oven for 3 hours to be mounted on glass slides
■ Olympus microscope with different magnifications used for morphological analysis

Question 14

Isolation of
nematodes

Answer 14

■ Larvae placed on white traps
■ First generation collected 2 to 4 days after infection
■ Second generation collected 5 to 7 days after emergence from dead larvae
■ EPNs rinsed with sterile water
■ Kept in 95% ethanol for molecular characterisation

Question 15

Scanning electron
microscopy (SEM)

Answer 15

■ 1 ml of EPNs from white traps placed in 1.5 ml Eppendorf tube
■ Heated at 80 ̊C for 5 minutes to kill them
■ Rinsed with Ringers solution
■ Fixed in 8% glutaraldehyde overnight
■ Dehydrated with 30, 50, 70, 90, 95 and 100% ethanol at 10 minute intervals
■ EPNs mounted on SEM stubs and critically point dried with CO2 for 2 hours
■ Coated with palladium or gold power
■ Scanned with FEI QUANTA SEM with digital camera

Question 16

Genomic DNA
extraction

Answer 16

■ One of each of the EPNs
■ Pure gene DNA purification kit
■ Stored at 4 ̊C

Question 17

PCR amplification

Answer 17

■ Conventional PCR used to amplify 18S rDNA sequences
Table 1: primers and corresponding sequences

■ Initial denaturation of 94 ̊C for 5 minutes, denaturation at
94 ̊C for 1 minute, annealing at 55 ̊C for 1 minute and
elongation at 72 ̊C for 2 minutes for 25 cycles and final
elongation at 72 ̊C for 10 minutes
■ PCR products confirmed with gel electrophoresis using 1% agarose gel
■ Amplicons sequenced and phylogenetically analysed