Multiplication of Entomopathogenic nematodes species

(Steinernema kraussie & Steinernema glaserie) on

different insect larvae.

KhanSa Irfan
2013-ag-3566
Department of Plant Pathology
University of Agriculture Faisalabad
 Entomopathogenic Nematodes

• The term Entomopathogenic originated from two Greek words “Entomon” which means

insect, and “pathogenic”, which means to causing disease.

• Entomopathogenic nematodes (EPNs) live parasitically inside the infected insect host, and so

they are endoparasites.

• Genera of Entomopathogenic nematode were first investigated in 1927.

Entomopathogenic nematodes are used in the biological control of harmful insects.
Insect parasitizing nematodes are classified into two families named as
1. Steinernematidae 2. Heterorhabditidae.

They both are associated with symbiotic bacteria and they form nematode-bacteria complex
(Boemare, 2002).

The both species Steinernematidae and Heterorhabditidae their infective juveniles produce
symbiotic bacteira in pro-intestine segment.
 Need for the project

Pleurotus ostreatus is an edible mushroom having excellent taste and Flavor.

During last few decades, the cultivation of Pleurotus ostreatus has been increased in various
developing countries like Pakistan.

Major part of the population of developing country like Pakistan has a problem of malnutrition and
cannot afford to eat meat.

The experiment was designed to decrease the problem of malnutrition by growing oyster
mushroom on fallen leaves of Ficus religiose tree along with different agricultural waste material.
 Objectives
• There is a big issue of chemical use as pesticide or insecticide on the crops to control the
different insect pests, which produced a lot of negative impact on the non-target specie, so
Entomopathogenic nematodes are use as bio-control agent to control the insect pest (Muller
and Gooch, 1982; Dhaliwal and Arora, 2001).

• To control the insect pest through EPNs

• To check the reproduction on different insect pest larvae

 Line of Work

Collection of different insect larvae

Mass culturing of EPNs
Galleria baiting technique
Isolation of infected juveniles from dead galleria
Identification of Entomopathogenic Nematode
Innoculation of EPNs on insect Larve
Counting of EPNs under stereoscope
 Collection of Insect Larvae

Different insect larvae is collected from the of vegetables and crops are collect from the field
area of Plant Pathology, square # 9, Square # 32 and vegetable area of university of
Agriculture, Faisalabad.
 Mass Culturing of EPNs

The spawn of pleurotus ostreatus was used in all the treatments containing different ratios of
dry weight of the substrate.

The spawn was delivered by the supervisor prepared in Mushroom Laboratory, Institute of
Horticultural Sciences.
 Procedure
10 g of spawn was used in each bag of substrate and plugged the mouth of bags with rubber
bands.

Bags were kept at room temperature (15°C to 20°C) and relative humidity maintained at 80-
90%.

The time was recorded in days for the development of 25%, 50%, 75% and 100% of
mycelial growth after spawning on all treatment bags containing Ficus religiosa leaves
amended with wheat straw, cotton waste, corn meal and thatch grass.
 Click icon to add picture

Watering
After the development of mycelia the bags start to
become white in color and holes were made in
bags.
Moisture level of the bags were observed visually
and was maintained by sprinkling water twice a
day.
 Data Recording

The data was recorded for following parameters i.e.

 Process of spawn running
Pinhead Development
 Formation of fruiting Bodies
Cropping yield of three flushes
Biological Efficiency
 Treatments

T1= 100% Ficus religiosa leaves

T2= 75% Ficus religiosa leaves + 25% Agri. waste material
T3= 50% Ficus religiosa leaves + 50% Agri. waste material
T4= 25% Ficus religiosa leaves + 75% Agri. waste material
T5= 0% Ficus religiosa leaves + 100% Agri. waste material
 Complete Randomized Deign was used to evaluate the results statistically
Results of 25% of mycelial growth
 Comparison of treatments means for 25%
achievement in fungal growth or spawn running
(Days).
Treatments (Ficus religiosa leaves with
Means (No. of Days)
agriculture waste material)

T1= Ficus leaves 100% 25.800 A

T2= Ficus leaves 75% + agri. waste 25% 23.200 B

T3= Ficus leaves 50% + agri. waste 50% 19.00 E

T4= Ficus leaves 25% + agri. waste 75% 21.200 D

T5= Ficus leaves 0% + agri. waste 100% 22.200 C

 25% Achievement of Mycelial Growth in bags
(Days)
30
25% mycelial growth (in Days)

25

20

15

10

0
T1 T2 T3 T4 T5

Treatments
Comparison of treatments means for 50% achievement
in fungal growth or spawn running (Days).

Treatments (Ficus religiosa leaves with agriculture

Means (No. of Days)
waste material)

T1= Ficus leaves 100% 28.600 A

T2= Ficus leaves 75% + agri. waste 25% 25.000 B

T3= Ficus leaves 50% + agri. waste 50% 21.800 C

T4= Ficus leaves 25% + agri. waste 75% 24.000 BC

T5= Ficus leaves 0% + agri. waste 100% 24.800 B

50% Achievement of Mycelial Growth in bags
(Days)
35

30
50% Mycelial Growth (Days)

25

20

15

10

0
T1 T2 T3 T4 T5

Treatments
 Comparison of treatments means for 75%
achievement in fungal growth or spawn
running (Days).
Treatments (Ficus religiose leaves with agriculture
Means (No. of Days)
waste material)

T1= Ficus leaves 100% 30.600 A

T2= Ficus leaves 75% + agri. waste 25% 27.800 AB

T3= Ficus leaves 50% + agri. waste 50% 24.200 C

T4= Ficus leaves 25% + agri. waste 75% 28.000 AB

T5= Ficus leaves 0% + agri. waste 100% 27.200 BC

75% Achievement of Mycelial Growth in bags
(Days)
35

30
75% Mycelial Growth (Days)

25

20

15

10

0
T1 T2 T3 T4 T5
Treatments
 Comparison of treatments means for 100%
achievement in fungal growth or spawn
running (Days).
Treatments (Ficus religiose leaves with agriculture
Means (No. of Days)
waste material)

T1= Ficus leaves 100% 35.400 A

T2= ficus leaves 75% + agri. waste 25% 31.600 B

T3= Ficus leaves 50% + agri. waste 50% 26.800 C

T4= Ficus leaves 25% + agri. waste 75% 28.800 BC

T5= Ficus leaves 0% + agri. waste 100% 30.200 B

100% Achievement of Mycelial Growth in bags
(Days)
40

35
100% Mycelial Grpwth (Days)

30

25

20

15

10

0
T1 T2 T3 T4 T5

Treatments
 Pinhead Formation

• The data was recorded in days taken for development of primordial formation in substrates.
comparison of treatments for the formation of
pinheads (In Days)

Treatments (Ficus religiose leaves with agriculture

Means (No. of Days)
waste material)

T1= Ficus leaves 100% 67.400 A

T2= Ficus leaves 75% + agri. waste 25% 62.000 B

T3= Ficus leaves 50% + agri. waste 50% 54.400 D

T4= Ficus leaves 25% + agri. waste 75% 57.600 C

T5= Ficus leaves 0% + agri. waste 100% 60.800 B

 Formation of Pinheads in Days
80

70
pinhead development(days)

60

50

40

30

20

10

0
T1 T2 T3 T4 T5
Treatments
 Comparison of treatments for the
development of numbers of pinheads.
Treatments (Ficus religiosa leaves with agriculture waste
Means (No. of Pinheads)
material)

T1= Ficus leaves 100% 36.000 C

T2= Ficus leaves 75% + agri. waste 25% 41.600 B

T3= Ficus leaves 50% + agri. waste 50% 47.200 A

T4= Ficus leaves 25% + agri. waste 75% 43.600 B

T5= Ficus leaves 0% + agri. waste 100% 42.600 B

 Number of Pinheads development:

60

50

40
No. of Pinheads

30

20

10

0
T1 T2 T3 T4 T5
Treatments
 Comparison of treatments for the
development of Fruiting Bodies (In Days)

Treatments (Ficus religiose leaves with agriculture

Means (No. of Days)
waste material)

T1= Ficus leaves 100% 21.200 A

T2= Ficus leaves 75% + agri. waste 25% 19.200 B

T3= Ficus leaves 50% + agri. waste 50% 9.000 E

T4= Ficus leaves 25% + agri. waste 75% 12.600 D

 Development of Fruiting Bodies (In Days):

25
Development of Fruiting body

20

15
(In Days)

10

0
T1 T2 T3 T4 T5
Treatments
 Comparison of treatments for the number of
flushes per bag:

Treatments (Ficus religiose leaves with agriculture

Means (No. of Flush per bag)
waste material)

T1= Ficus leaves 100% 2.0000 C

T2= Ficus leaves 75% + agri. waste 25% 3.2000 C

T3= Ficus leaves 50% + agri. waste 50% 8.2000 A

T4= Ficus leaves 25% + agri. waste 75% 7.4000 A

T5= Ficus leaves 0% + agri. waste 100% 5.4000 B

Relative Development of Number of flushes per
bag:
9

8
Number of Flush per bag

0
T1 T2 T3 T4 T5
Treatments
Total yield
Comparison of treatments for the Total Yield (g):

Treatments (Ficus religiosa leaves with agriculture

Means (Grams)
waste material)

T1= Ficus leaves 100% 250.49 E

T2= Ficus leaves 75% + agri. waste 25% 292.64 D

T3= Ficus leaves 50% + agri. waste 50% 413.48 A

T4= Ficus leaves 25% + agri. waste 75% 376.65 B

T5= Ficus leaves 0% + agri. waste 100% 340.91 C

 Total Yield (g)
500

450

400

350
Total Yield (g)

300

250

200

150

100

50

0
T1 T2 Treatments
T3 T4 T5
 Biological Efficiency

• Biological efficiency was determined through the formula.

 Comparison of treatments for the Biological
efficiency of mushroom.
Treatments (Ficus religiose leaves with agriculture waste
Means(%)
material)

T1= Ficus leaves 100% 50.099 E

T2= Ficus leaves 75% + agri. waste 25% 58.526 D

T3= Ficus leaves 50% + agri. waste 50% 82.702 A

T4= Ficus leaves 25% + agri. waste 75% 75.329 B

T5= Ficus leaves 0% + agri. waste 100% 68.183 C

 Biological Efficiency

90

80
Biological Efficiency (%)

70

60

50

40

30

20

10

0
T1 T2 T3 T4 T5

Treatment
 Summary

The study was carried out in the Mushroom laboratory, Institute of Horticultural Sciences, UAF to
investigate the effect of on mycelial growth and fructification of Pleurotus ostreatus on ficus religiosa
leaves along with agri. waste material.

As a whole pattern of spawning phases of 25%, 50%, 75% and 100% completion on ficus religiosa
leaves contained agri. waste fifty-fifty substrate was significant and the least significantly effective
substrate supplemented with 100% ficus religiose.
As far as yield performance of oyster mushroom in three flushes were concerned, the significantly
effective substrate was simple treatment 3 which produced the maximum yield production followed
by other supplementary materials. Treatment T3= 50% Ficus leaves + 5% Agri. waste gave better yield
as compared to others.

Data regarding the biological efficiency of Oyster mushroom on Ficus religiosa with some
supplements was recorded. The maximum and minimum biological efficiency was obtained on
Treatment T3= 50% Ficus leaves + 5% Agri. waste and T1= Ficus leaves 100% supplemented with rice
bran respectively.
THANK YOU….!