Influence of soil substrate on the biocontrol
capacity of Pseudomonas CMR12a against
Rhizoctonia root rot on bean
Hua, H. G. K., D´aes, J., De Maeyer, K. and Höfte, M.
Outline

Introduction

Methodology

Results and Discussion

Conclusions

Future prospectives
Introduction
Beans (Phaseolus vulgaris L.)
•
Most important grain legumes for direct human consumption
in the world.
Introduction
Beans (Phaseolus vulgaris L.)
•
Most important grain legumes for direct human consumption
in the world.
• Health benefits from consuming beans on a regular basis:

Maintenance of a healthy weight

Reduced risk of diabetes

Reduced risk of heart disease

Reduced risk of colon cancer
Introduction
Rhizoctonia solani
• Very common soil-borne pathogen
Introduction
Rhizoctonia solani
• Very common soil-borne pathogen
• Exists primarily as

Mycelium

Sclerotia
Introduction
Rhizoctonia solani
• Very common soil-borne pathogen
• Exists primarily as

Mycelium

Sclerotia
• Great diversity of host plants
Introduction
Root rot on Bean
• Occur on young seedlings
•
Small, oval to elliptical,
reddish-brown sunken lesions
or cankers on stem and roots
•
Severely infected seedlings = dead
Research goal
Determining influence of different soil-sand mixtures on
biocontrol capacity of Pseudomonas CMR12a against
Rhizoctonia root rot
Introduction
• Biocontrol agents:

Pseudomonas CMR12a

CMR12a-mutants
???
Introduction
• Biocontrol agents:

Pseudomonas CMR12a

CMR12a-mutants
• Background knowledge:

Non-pathogenic on bean

Able to produce important antibiotics:


Phenazines (Phz)

Cyclic lipopeptides (CLPs)
Successful biocontrol agent
In vivo biocontrol of Rhizoctonia root rot on bean
• Soil substrates:

25% potting soil : 75% sand

50% potting soil : 50% sand

75% potting soil : 25% sand
In vivo biocontrol of Rhizoctonia root rot on bean
• Soil substrates:

25% potting soil : 75% sand

50% potting soil : 50% sand

75% potting soil : 25% sand
• Fungal isolate:

AG 2-2 18
In vivo biocontrol of Rhizoctonia root rot on bean
• Soil substrates:

25% potting soil : 75% sand

50% potting soil : 50% sand

75% potting soil : 25% sand
• Fungal isolate:

AG 2-2 18
• Bacterial isolates:

CMR12a (Phz+ and CLP1+)

CMR12a-∆Phz (Phz- and CLP1+)

CMR12a-CLP1 (Phz+ and CLP1-)

CMR12a- ∆Phz-CLP1 (Phz- and CLP1-)
In vivo biocontrol of Rhizoctonia root rot on bean
Day 0
Preparation of fungal inoculum
In vivo biocontrol of Rhizoctonia root rot on bean
Day 0
Preparation of fungal inoculum
Day 3
Seed germination
In vivo biocontrol of Rhizoctonia root rot on bean
Day 0
Preparation of fungal inoculum
Day 3
Seed germination
Bacterial application (106 CFU g-1 soil)
Day 6
Seed sowing
In vivo biocontrol of Rhizoctonia root rot on bean
Day 0
Preparation of fungal inoculum
Day 3
Seed germination
Bacterial application (106 CFU g-1 soil)
Day 6
Day 9
Seed sowing
Fungal inoculation (4 infected kernels/seedling)
In vivo biocontrol of Rhizoctonia root rot on bean
Day 0
Preparation of fungal inoculum
Day 3
Seed germination
Bacterial application (106 CFU g-1 soil)
Day 6
Day 9
Day 15
Seed sowing
Fungal inoculation (4 infected kernels/seedling)
Evaluation
In vivo biocontrol of Rhizoctonia root rot on bean
Class 0: Healthy, no symptoms observed
1: Lesion ≤ 25% of stem and/or hypocotyl
2: Lesion ≤ 50% of stem and/or hypocotyl
3: Lesion ≤ 75% of stem or hypocotyl
4: Hypocotyl is completely decayed and seedling dead
0
1
2
3
4
In vivo biocontrol of Rhizoctonia root rot on bean
3.0
Disease severity
2.5
f
f
ef
ef
ef ef
2.0
de
25% ps
de
50% ps
1.5
bc
bc
1.0
75% ps
cd
bcd
bc
bc
b
0.5
0.0
AG 2-2 18
CMR12a
∆Phz
CLP1
∆Phz-CLP1
Phz
+
-
+
-
CLP1
+
+
-
-
In vivo biocontrol of Rhizoctonia root rot on bean
3.0
Disease severity
2.5
f
f
ef
ef
ef ef
2.0
de
25% ps
de
50% ps
1.5
bc
bc
1.0
75% ps
cd
bcd
bc
bc
b
0.5
0.0
AG 2-2 18
CMR12a
∆Phz
CLP1
∆Phz-CLP1
Phz
+
-
+
-
CLP1
+
+
-
-
Influence of soil substrates on the growth of bean seedlings
Day 0
•
Seed sowing
•
Soil substrates used:

25% potting soil: 75% sand

50% potting soil: 50% sand

75% potting soil: 25% sand
Influence of soil substrates on the growth of bean seedlings
Day 0
•
Seed sowing
•
Soil substrates used:
Day 6, 9 and 15
Data record:

Diameter of hypocotyl

25% potting soil: 75% sand

Length of shoot and root

50% potting soil: 50% sand

Fresh and dry weight of shoot and root

75% potting soil: 25% sand
Influence of soil substrates on the growth of bean seedlings
Fresh weight of Root
Fresh weight of Shoot
2.5
7.0
25% ps
50% ps
25% ps
75% ps
a
50% ps
a
75% ps
6.0
b
b
a
1.5
c
bc
1.0
c
a
a
a
a
5.0
a
4.0
c
3.0
a
0.5
Weight (g)
Weight (g)
2.0
2.0
a
1.0
a
a
b
a
b
b
a
a
0.0
0.0
6
9
Days post sowing
12
15
6
9
12
Days post sowing
15
Influence of soil substrates on the growth of bean seedlings
Fresh weight of Root
Fresh weight of Shoot
2.5
7.0
25% ps
50% ps
25% ps
75% ps
a
50% ps
a
75% ps
6.0
b
b
a
1.5
c
bc
1.0
c
a
a
a
a
5.0
a
4.0
c
3.0
a
0.5
Weight (g)
Weight (g)
2.0
2.0
a
1.0
a
a
b
a
b
b
a
a
0.0
0.0
6
9
Days post sowing
12
15
6
9
12
15
Days post sowing
•
Optimal growth was observed in substrate containing 75% potting soil
•
The decrease in proportion of potting soil results in the decrease in seedling growth
25%
50%
75%
Fig 1. Difference in the development of bean seedlings grown on substrates
containing various percentage of potting soil
Influence of soil substrates on the spread of Rhizoctonia solani
Day 0
Preparation of fungal inoculum
Influence of soil substrates on the spread of Rhizoctonia solani
Day 0
Preparation of fungal inoculum
Day 3
Seed germination
Influence of soil substrates on the spread of Rhizoctonia solani
Day 0
Preparation of fungal inoculum
Day 3
Seed germination
Seed sowing
Day 6
Fungal inoculation (1 infected kernel/seedling)
Influence of soil substrates on the spread of Rhizoctonia solani
Day 0
Preparation of fungal inoculum
Day 3
Seed germination
Seed sowing
Day 6
Day 10, 13, 16 and 19
Fungal inoculation (1 infected kernel/seedling)
Inserting sterile toothpicks along the seedling rows
Influence of soil substrates on the spread of Rhizoctonia solani
Day 0
Preparation of fungal inoculum
Day 3
Seed germination
Seed sowing
Day 6
Day 10, 13, 16 and 19
Fungal inoculation (1 infected kernel/seedling)
Inserting sterile toothpicks along the seedling rows
Removing the toothpicks from soil
Day 12, 15, 18 and 21
Placing toothpicks on Rhizoctonia selective medium
Determining the number of toothpicks colonized by
Rhizoctonia hyphae
Fig 2. Correspondence between the colonization of R. solani on toothpicks and the
appearance of disease symptoms on bean seedlings
Influence of soil substrates on the spread of Rhizoctonia solani
Sterile soil
Non‐sterile soil
120
25% ps
50% ps
75% ps
100
80
a
a
a
60
a
a
a
a
a
a
40
20
a
a
a
0
% toothpicks colonized by Rhizoctonia
% toothpicks colonized by Rhizoctonia
120
25% ps
9 dpi
12 dpi
Days post inoculation
15 dpi
75% ps
a
a
a
a
80
60
a
a
a
40
b
b
a
20
0
6 dpi
50% ps
100
a
b
6 dpi
9 dpi
12 dpi
Days post inoculation
•
Sterile soil: no significant difference amongst treatments
•
Non-sterile soil: fastest invasion was observed in substrate with 25% ps
15 dpi
Influence of soil substrates on the spread of Rhizoctonia solani
120
25% ps ‐ SS
25% ps ‐ NS
100
a
a
80
a
a
a
60
a
40
20
a
a
0
6 dpi
9 dpi
12 dpi
15 dpi
Days post inoculation
% toothpicks colonized by R. solani
% toothpicks colonized by R. solani
120
50% ps ‐ SS
50% ps ‐ NS
100
a
a
a
80
a
60
b
40
b
20
a
0
b
6 dpi
9 dpi
12 dpi
15 dpi
Days post inoculation
% toothpicks colonized by Rhizoctonia
120
75% ps ‐ SS
75% ps ‐ NS
100
a
a
a
80
a
60
a
40
20
Rhizoctonia hyphae spreaded
b
slower in non‐sterile soil
a
a
0
6 dpi
9 dpi
12 dpi
Days post inoculation
15 dpi
Influence of soil substrates on the spread of Rhizoctonia solani
4.0
d
d
3.5
cd
cd
bcd
3.0
abc
Disease severity
2.5
ab
a
2.0
Sterile soil
1.5
Non‐sterile soil
1.0
0.5
0.0
25% ps
50% ps
Treatments
75% ps
100% ps
Influence of soil substrates on the spread of Rhizoctonia solani
4.0
d
d
3.5
cd
cd
bcd
3.0
abc
ab
Disease severity
2.5
a
2.0
Sterile soil
1.5
Non‐sterile soil
1.0
0.5
0.0
25% ps
50% ps
75% ps
100% ps
Treatments
•
Sterile soil: no significant difference amongst soils
Influence of soil substrates on the spread of Rhizoctonia solani
4.0
d
d
3.5
cd
cd
bcd
3.0
abc
ab
Disease severity
2.5
a
2.0
Sterile soil
1.5
Non‐sterile soil
1.0
0.5
0.0
25% ps
50% ps
75% ps
100% ps
Treatments
•
Sterile soil: no significant difference amongst soils
•
Non-sterile soil: disease severity was highest in 25% ps and lowest in 50% ps
Increase in percentage
of sand present
Increase in percentage
of sand present
Increase in
the spread of
Rhizoctonia
hyphae
Decrease in
the growth of
seedlings
Increase in percentage
of sand present
Increase in
Decrease in
the spread of
the growth of
Rhizoctonia
seedlings
hyphae
Increase in disease
severity
Increase in percentage
of sand present
Increase in
Decrease in
the spread of
the growth of
Rhizoctonia
seedlings
hyphae
Increase in disease
severity
Increase in percentage
of sand present
75% potting soil
Phz or CLPs alone is
sufficient
Increase in
Decrease in
the spread of
the growth of
Rhizoctonia
seedlings
hyphae
Increase in disease
severity
50% potting soil
25% potting soil
Increase in percentage
of sand present
75% potting soil
Phz or CLPs alone is
sufficient
Increase in
the spread of
Rhizoctonia
Decrease in
the growth of
seedlings
hyphae
Increase in disease
severity
50% potting soil
25% potting soil
Phz and CLPs are
required to be
effective
Future Prospects
• Studying the survival and multiplication capacity of CMR12a and
CMR12a-mutants in different soil substrates
• Analysing the physical and chemical characteristics of soil combinations
used.
• Exploring induced systemic resistance capacity of phenazines and
cyclic lipopeptides
Acknowledgements
• Prof. Monica Höfte
• Katrien De Maeyer
• Jolien D’aes
• Sarah Van Beneden
• Soraya De Carvalho França
• Ilse Delaere
• “Special Research Fund” (BOF) Ghent University