Rufus seminar 2017_mechanisms of biological control of plant diseases edited_09_24

MECHANISMS OF ACTION OF
NEMATICIDAL BACTERIA
Student: Rufus Akinrinlola
Date: September 25, 2017
Acknowledgement
Advisors;
Gary Yuen
Tony Adesemoye
Yuen Team

 Topics
 Why this topic?
 Presentation objectives
 Impacts of plant parasitic nematodes (PPNs)
 Control strategies for PPN diseases
 Biocontrol agents of PPNs
 Classes of nematicidal bacteria
 Examples of Nematicidal bacteria
 Biocontrol potentials of some nematicidal
 Summary

 Why this topic?
https://ohioline.osu.edu/factsheet/plpath-gen-8
 My thesis work focus on PGPR
 Why discussing NEMATICIDAL BACTERIA ?
 Personal curiosity
 Personal interest in biocontrol and plant parasitic
nematodes

 Presentation objectives
Three Objectives
 To show some of the different mechanisms of action of
Nematicidal bacterial.
 Show how these mechanisms work.
 Show how the understanding of the different mechanisms
affects the development of a potential nematicidal biocontrol
agent.

 Impacts of plant parasitic nematodes (PPNs)
 Global threat to crop production
 Annual crop damage; $157 billion USD
 Attack up to 4,100 plant species
ucdavis.edu/nemaplex/images

 Control strategies for PPN diseases
 Host Resistance
Limited due to high genetic diversity among nematode populations
 Cultural Practices
PNNs can attack almost all plants
They can survive in soils for many years
 Chemical application
Are toxic to wildlife and human health
 Biological control
Remains potential alternative/sustainable approach

 Biocontrol agents of PPNs
Nematophagous
fungi
Biocontrol
agents of
PPNs
Nematicidal
bacteria

 Classes of nematicidal bacteria
 Parasitic bacteria
 Obligate parasite
 Opportunistic parasite
 Non-parasitic rhizobacteria
 Rhizobacteria
 Parasporal Cry protein-forming bacteria
 Endophytic bacteria
 Symbiotic bacteria
Tian, B., Yang, J., & Zhang, K. Q. (2007). Bacteria used in the biological control of plant-parasitic nematodes: populations, mechanisms of action, and future prospects. FEMS microbiology ecology, 61(2), 197-213.

 Mechanisms of action of
nematicidal bacteria
 Parasitism;
 Toxins or enzymes activity
 Interfering with host
recognition
 Competing for nutrients
 Induced systemic resistance
 Promoting plant health

 Pasteuria penetrans
 Bacillus thuringiensis (Bt) Toxins
 Bacillus firmus DS-1
 Bacillus nematocida B16
 Lyzobacter enzymogenes C3
 Examples of Nematicidal bacteria

 Pasteuria penetrans
 An obligate parasite of nematode
 Mycelial and endospore-forming bacterium
 Colonize up to 300 nematode species
Siddiqui and Mahmood, 1999; Tian et al., 2007
https://smartsite.ucdavis.edu/access/content/user/00002950/courses/slides/fromWWW/manage/pasteu
r.gif
Mankau et al., 1976).

 Pasteuria penetrans mechanisms
Formation and proliferation of vegetative
microcolonies within female.
Breakdown of reproductive system of female
nematode and release of mature endospores
Siddiqui and Mahmood, 1999; Tian et al., 2007
As nematode move through soil, endospore
attaches to cuticles of J2 nematode
Germination of spore and germ tube
penetration of cuticle follows
1
2
3

Li, J., Zou, C., Xu, J., Ji, X., Niu, X., Yang, J., ... & Zhang, K. Q. (2015). Molecular mechanisms of nematode-nematophagous microbe
interactions: basis for biological control of plant-parasitic nematodes. Annual review of phytopathology, 53, 67-95.
 Bacillus thuringiensis mechanisms
 Produce a proteinaceous
substance known as
protoxin crystals (Bt toxin)
 BT toxins form pores
within nematodes and lyses
their intestine
 Nematode death
BT Toxin
http://curiosidadesdelamicrobiologia.blogspot.com

Method:
C. elegans was fed with genetically transformed E.coli
with or without a specific BT toxin gene, Cry5B
E. coli without
Cry5B
E. Coli with Full-
length Cry5B
E. Coli with
truncated Cry5B
Li, J., Zou, C., Xu, J., Ji, X., Niu, X., Yang, J., ... & Zhang, K. Q. (2015). Molecular mechanisms of nematode-nematophagous microbe interactions: basis for biological control of plant-parasitic nematodes. Annual
review of phytopathology, 53, 67-95.
Effects of BT toxin on free-living nematode; C. elegans

1
Result:
E. coli without
Cry5B
E. Coli with Full-
length Cry5B
E. Coli with
truncated Cry5B
Li, J., Zou, C., Xu, J., Ji, X., Niu, X., Yang, J., ... & Zhang, K. Q. (2015). Molecular mechanisms of nematode-nematophagous microbe interactions: basis for biological control of plant-parasitic nematodes. Annual
review of phytopathology, 53, 67-95.
BT toxin was lethal to C. elegans after ingestion.
Cry5B exerts its effects on the gut epithelial cell membrane,
forming pores and subsequent lysis and death of the nematodes

 Bacillus firmus DS-1 mechanisms
 DS-1 provides excellent control of PPNs
 A commercial nematicidal bacterial
 Produce a novel serine protein known as Sep1
 The protein can degrade cuticle-associated protein and
nematode physical barriers
 Highly toxic against root-knot and soybean cyst
nematodes
Geng et al., 2016).

Method
1. Cyst nematode and root-knot nematodes were exposed to
different levels of Bacillus firmus supernatant for 3 days
2. Lethality of supernatant against the PPNs were determined
Effects of Strain DS-1 on cyst and root-knot nematodes
Geng et al., 2016).

Results
92.54% - 97.81%
mortality rate
Geng et al., 2016).

Geng et al., 2016).
Results
59.5% to 70%
Mortality rate

Method and Result
Identifying the nematicidal virulence factors in strain DS-1
1. Whole genome sequence and analysis were conducted based on
several reported virulence genes.
2. Results showed that B. firmus DS-1 genome harbors multiple
potential extracellular protease genes
3. To assess the nematicidal activity of these genes, 13 genes were
cloned and expressed in E. coli
Geng et al., 2016).

Method and Result
4. Nematicidal bioassay showed that one of the proteins
(EWG10090) expressed from E. coli, was lethal against C.
elegans N2 (73.2% mortality).
5. The protein was selected for further study and designated as
Serine Protease 1 (Sep1)
Geng et al., 2016).
Identifying the nematicidal virulence factors in strain DS-1

Geng et al., 2016).
Method:
 Different doses of E. coli-purified Sep1 proteins exposed to M.
incognita J2 animals.
To test the nematocidal activities of Sep1 against PPNs

Geng et al., 2016).
Method:
 Different doses of E. coli-purified Sep1 proteins exposed to M.
Results:
 The results showed that Sep1 caused significant lethality to M.
 Nematode mortality increased gradually with every increase in
Sep1 concentration.
To test the nematocidal activities of Sep1 against PPNs

Result:
Geng et al., 2016).
Nematicidal activities of Sep1 against PPNs
Sep1 caused significant lethality to M. incognita J2 animals.

Method and Results
Effects of Sep1 on intestinal structures of C. elegans and M.
incognita
1. J2 animals of M. incognita were fed with Sep1 for 48 h
2. Control JS animals were fed with resorcinol (RES) solution.
3. The treatments were observed with optical microscope.

Method and Results
Effects of Sep1 on intestinal structures of C. elegans and M.
incognita
1. J2 animals of M. incognita were fed with resorcinol (RES) for 48
2. Control JS animals were fed with resorcinol (RES) solution.
3. The treatments were observed with optical microscope.
 The results demonstrated that the Sep1 protein damage M.
incognita tissues.

Damaged esophageal
balloon and stylet
The results demonstrated that the Sep1
protein damage M. incognita tissues.

Damaged
intestinal tissues
No Toxin Sep 1
The results demonstrated that the Sep1
protein damage M. incognita tissues.

 Bacillus nematocida B16
Trojan horse mechanisms
 An opportunistic parasitic bacteria
 Exhibits Trojan horse mechanism;
 Secretion of volatile organic compounds to lure nematodes
 Secretion of two extracellular proteases inside the nematode
 Alkaline serine protease Bace16
 Neutral protease Bae16
 Proteases kill nematode by destroying the intestine
Niu, Q., Huang, X., Zhang, L., Xu, J., Yang, D., Wei, K., ... & Yang, J. (2010). A Trojan horse mechanism of bacterial pathogenesis against
nematodes. Proceedings of the National Academy of Sciences, 107(38), 16631-16636.

Effects of volatile organic compounds produced by B.
Nematocida on C. elegans
Method:
Two petri plates
containng C. elegans
nematodes

Method:
Two petri plates
nematodes
Inversion of B.
nematoda plate on
first C. elegans
nematode plate

Method:
Two petri plates
nematodes
Inversion of B.
nematoda plate on
first C. elegans
nematode plate
Inversion of E. coli
plate on second C.
elegans nematode
plate

Result:

Results
56% of C. elegans
migrated toward the B.
nematocida lawn.
within 8 h.
Only 12% of C.
elegans moved toward
the control ( E. coli)
lawn.
280
B. Nematoda
plate
Control (E.
coli) plate)
60

1. C. elegans was exposed to four different treatments including ;
 B. nematoda (antibiotic-resistant) mutant
 Wild type B. nematoda
 E.coli
 Blank plate without any bacterial cell
2. Worms were selected from each treatment, thoroughly grounded
and plated onto LB medium contain antibiotic.
Effects of Bacillus nematocida B16 on C. elegans
Method:

Results:
 Antibiotic-resistant bacterial colonies were observed from
dead worms infected by the mutant strain
 No colonies were recovered from the controls
 The mutant and wild-type strains caused extensive intestinal
damage.
 Worms that swallowed E. coli cells had no damage to their
intestinal tracts
Effects of Bacillus nematocida B16 on C. elegans

Niu, Q., Huang, X., Zhang, L., Xu, J., Yang, D., Wei, K., ... & Yang, J. (2010). A Trojan horse mechanism of bacterial pathogenesis against nematodes. Proceedings of the National Academy of Sciences, 107(38), 16631-
16636.
C. elegans treated with B.
nematoda shows Lightly
exfoliated cuticle
Control : C. elegans treated with
E. coli with undisturbed surface
with smooth cuticle structure
damage.
Results:

Niu, Q., Huang, X., Zhang, L., Xu, J., Yang, D., Wei, K., ... & Yang, J. (2010). A Trojan horse mechanism of bacterial pathogenesis against nematodes. Proceedings of
the National Academy of Sciences, 107(38), 16631-16636.
C. elegans treated with B.
nematoda shows extensive
damaged of intestinal structures
Control : C. elegans treated
with E. coli with intact
intestine and cuticle
Results:
damage.

 Lyzobacter enzymogenes C3
mechanisms
Yuen, 2006; Chen et al.,2006
 C3, is a broad spectrum biocontrol bacteria .
 Involves two modes of action;
 HSAF production
 Chitinase enzyme activity
 Suppress PPN diseases in soil systems
 Increase plant biomass

Method:
1. C. elegans nematodes were placed into C3 culture.
2. Control nematodes were placed in medium cultures of E. coli
3. Nematode development, reproduction, and survival were daily
observed.
4. C3 cells populations were also determined.
Chen et al.,2006
Effects of C3 on reproduction and survival of C. elegans

Method:
1. C. elegans nematodes were placed into C3 culture.
2. Control Nematodes were placed in medium cultures of E. coli
3. Nematode development, reproduction, and survival were daily
observed.
4. C3 cells populations were also determined.
Result
 C3 reduced the reproduction and survival of C. elegans
 C3 cells population increases in C. elegans culture plate
Chen et al.,2006
Effects of C3 on reproduction and survival of C. elegans

Result:
Very few nematode eggs
and juveniles remaining in
C3 culture plate after day 1
More nematode eggs and
juveniles in E.coli plate after
1 day
Chen et al.,2006

At day 2 and 3, no nematode eggs
left and only a few juveniles
remaining in C3 culture
Eggs and juveniles of nematode
were to many to count in E.coli
plate after at after day 2
Chen et al.,2006
Result:

No adult nematode left in C3
culture after day 2
Adult nematodes present in
E.coli plate after day 2
Chen et al.,2006
Result:

Result
 C3 cells population increases in C. elegans culture plate
C3 population greatly
increased in culture plate
containing C. elegans
Chen et al.,2006

nematode adults less or
no progeny/dish on C3
plate compared up to 68
progeny on control plate
Chen et al.,2006
Result
 C3 reduced adults C. elegans in culture plate

Effect of C3 on survivability of Meloidogyne javanica (Root-
knot nematode) juveniles
 M. javanica were exposed C3 broth from 7 d old culture, with or
without subsequent storage for 16 d at 4°C.
Method
Chen et al.,2006

Effect of C3 on survivability Meloidogyne javanica (Root-knot
nematode) juveniles
Result
 M. javanica were exposed C3 broth from 7 d old culture, with or
without subsequent storage for 16 d at 4°C.
Method
 No M. javanica survived after 4 d exposure to both
C3 treatments
Chen et al.,2006

Effects of C3 on various plant-parasitic nematodes
Result
 PPNs were exposed to C3 culture, C3 filtrate, and controls without C3
 PPNs were counted daily and scored as active, inactive, or missing
Method
 C3 caused adults and juveniles of PPNs to become inactive and
then dissolve
Chen et al.,2006

Chen et al.,2006
Number of nematodes that were not disintegrated after
exposure to different treatments for 2 d
 C3 caused disintegration of more adults and
juveniles of PPNs compared to the controls
Pratylenchus penetransAphelenchoides fragariae
Result
 C3 caused adults and juveniles of PPNs to become inactive and
then dissolve

Chen et al.,2006
 Pratylenchus penetrans in C3 for 2 d
Body of Pratylenchus penetrans, as
dissolved by C3 in 48 h

Effects of C3 HSAF on nematodes
Yuen et al., (2006).
Method:
1. HSAF (dihydromaltophilin) was extracted from C3 culture
2. HSAF added to E coli culture medium plate
3. Ten C. elegans adults were added to the medium
4. Control medium plate had no HSAF
5. The nematodes activity were monitored for 48 h

Effects of C3 HSAF on nematodes
Method:
1. HSAF (dihydromaltophilin) was extracted from C3 culture
2. HSAF added to E coli culture medium plate
3. Ten C. elegans adults were added to the medium
4. Control medium plate had no HSAF
5. The nematodes activity were monitored for 48 h
Results:
 HSAF caused rapid inactivation of adult nematodes

Results:
 C3 HSAF caused rapid inactivation of adult nematodes
Fewer than 40% of the
nematodes exposed to
HSAF were active in 24
h. Over 90% of
nematodes remained
active in the control

Results:
 HSAF-minus mutant K19 failed to inactivate nematodes
Mutants failed to
inactivate nematodes
Wildtype C3 eliminated
nematodes from plate
A proof that HSAF is responsible
for rapid inactivation of nematodes
by Wildtype C3

Biocontrol potentials of some
nematicidal bacteria in soil system
experiments

Biocontrol potentials of Pasteuria penetrans
Control of M. incognita on cucumber by
Pasteuria penetrans in greenhouse and
microplots experiments
(Kokalis-Burelle, 2015).

Method:
1 3 4 52
 Soil mix containing 1 mL of nematode egg suspension
(1000 eggs/mL) was placed into 5 pots.

Method:
Steamed
soilControl
106 spores
per seed
1.5x 105
endospores
after planting
106 spores per
seed + 1.5x 105
after planting
C P1 P2 P3S
 Appropriately treated/transplants planted into the nematode
infested potted soil for 8 to 10 weeks
 All plants evaluated for different parameters

Method:
Telone II
FumigatedControl
106 spores
per seed
1.5x 105
endospores
after planting
106 spores per
seed + 1.5x 105
after planting
 Appropriately treated/transplants planted into the nematode
infested potted soil for 8 to 10 weeks
 All plants evaluated for different parameters
C P1 P2 P3S

microplots experiment
Greenhouse study results

microplots experiment
Microplots study results
Only root weight increase, occurred in one treatment (1.5x 105
endospores after planting)
Telone II fumigation (control) reduced more galls than other
treatments.

Yuen et al.,
unpublished
Control of cyst nematodes on cabbage,
sugarbeet and soybean by strain C3 and the role
of the antibiotic HSAF in the biological control
activity in soil systems
Method
 Strain C3 was applied to the roots of cabbage, sugarbeet, and
soybean grown in growth pouches, sand, and a sand-soil medium
 Cabbage and sugarbeet roots were challenged with SBCN, while
soybean roots were inoculated with SCN.

Control of cyst nematodes on cabbage,
sugarbeet and soybean by Lysobacter
enzymogenes strain C3
Results:
 C3 reduced the number of SBCN nematodes on sugarbeet roots
compared to the control
 C3 reduced numbers of SBCN cysts and eggs on cabbage roots
compared to the control.
 C3 inhibited SCN egg production relative to the control on
soybean
Yuen et al.,
unpublished

Results:
 C3 reduced SBCN nematodes on sugarbeet roots compared to
the control
Yuen et al.,
unpublished

Results:
 C3 reduced numbers of SBCN cysts and eggs on cabbage roots
compared to the control.
Yuen et al.,
unpublished
Treatment Cysts per plant Total eggs & J2 Eggs & J2 per cyst Shoot dry wt. (g)
Cabbage alone - - - 0.44
C3 alone - - - 0.40
SCBN alone 52 8445 140 0.45
SCBN + C3 37 4734 106 0.40
P- value 0.051 0.009 0.143 Not significant

Results:
 C3 reduced SCN egg production relative to the control on
soybean
Trial Treatment Eggs per
plant
Eggs per g
root
Root wt. (g) Shoot wt. (g)
1 C3 + SCN 43 72 0.61 B 4.61
SCN only 136 200 0.76 B 4.72
C3 only - - 0.78 B 4.74
No treatment - - 1.17 A 4.84
P <0.001 0.326 0.005 NS
2 C3 + SCN 140 1164 0.11 No data
SCN only 1090 6331 0.05 No data
C3 only - - 0.04 No data
No treatment - - 0.11 No data
P <0.001 <0.001 0.369 -
3 C3 + SCN 477 4387 0.12 C 1.13
SCN only 1763 14360 0.12 C 0.99
C3 only - - 0.24 B 1.12
No treatment - - 0.34 A 1.29 Yuen et al.,
unpublished

Results:
 HSAF-minus mutant K19 failed to suppress SCN on soybean
Yuen et al.,
unpublished
Trial Treatment Eggs per plant Eggs per g root Root wt. (g) Shoot wt. (g)
1 C3 + SCN 599 B 1542 C 0.41 B 1.75
K19 + SCN 1710 A 6442 A 0.29 B 1.44
SCN only 1023 A 2487 B 0.38 B 1.71
C3 only - - 0.57 A 1.63
K19 only - - 0.57 A 1.39
P 0.001 <0.001 <0.001 NS
2 C3 + SCN 75 B 121 B 0.63 bc 1.74 A
K19 + SCN 141 A 258 A 0.55 c 1.42 BC
SCN only 166 A 314 A 0.59 bc 1.31 BC
C3 only - - 0.79 a 1.53 AB
K19 only - - 0.68 ab 1.21 C
No treatment - - 0.69 ab 1.41 BC
P 0.034 0.034 0.084 0.029
3 C3 + SCN 436 b 1379 B .31 1.13
K19 + SCN 820 a 2506 A .33 1.22
SCN only 847 a 3058 A .28 1.03
C3 only - - .31 1.24
K19 only - - .36 1.26
No treatment - - .34 1.17

Trial Treatment Eggs per plant Eggs per g root Root wt. (g) Shoot wt. (g)
1 C3 + SCN 599 B 1542 C 0.41 B 1.75
K19 + SCN 1710 A 6442 A 0.29 B 1.44
SCN only 1023 A 2487 B 0.38 B 1.71
C3 only - - 0.57 A 1.63
K19 only - - 0.57 A 1.39
P 0.001 <0.001 <0.001 NS
2 C3 + SCN 75 B 121 B 0.63 bc 1.74 A
K19 + SCN 141 A 258 A 0.55 c 1.42 BC
SCN only 166 A 314 A 0.59 bc 1.31 BC
C3 only - - 0.79 a 1.53 AB
K19 only - - 0.68 ab 1.21 C
No treatment - - 0.69 ab 1.41 BC
P 0.034 0.034 0.084 0.029
3 C3 + SCN 436 b 1379 B .31 1.13
K19 + SCN 820 a 2506 A .33 1.22
SCN only 847 a 3058 A .28 1.03
C3 only - - .31 1.24
K19 only - - .36 1.26
No treatment - - .34 1.17
P 0.059 0.028 0.445 NS
Results:
 HSAF-minus mutant K19 failed to suppress SCN on soybean
Yuen et al.,
unpublished
A proof that HSAF is
responsible for the
suppression of SCN
in Wildtype C3
treated soybean
plant

 Summary
 PPNs diseases posses major threat to global crop production
 Biological control with nematicidal bacteria is gaining more
attention in controlling PPNs
 Major mechanisms of nematicidal bacterial include parasitism;
toxins and enzymes activities
 Pasteuria penetrans, Bacillus firmus, and Lyzobacter enzymogenes
have shown successful control of several PPN diseases
 More nematicidal bacterial would be commercialized as we gain
more understanding

 Li, J., Zou, C., Xu, J., Ji, X., Niu, X., Yang, J., ... & Zhang, K. Q. (2015). Molecular mechanisms of
nematode-nematophagous microbe interactions: basis for biological control of plant-parasitic
nematodes. Annual review of phytopathology, 53, 67-95.
 Tian, B., Yang, J., & Zhang, K. Q. (2007). Bacteria used in the biological control of plant-parasitic
nematodes: populations, mechanisms of action, and future prospects. FEMS microbiology
ecology, 61(2), 197-213.
 Siddiqui, Z. A., & Mahmood, I. (1999). Role of bacteria in the management of plant parasitic nematodes:
a review. Bioresource Technology, 69(2), 167-179.
 Mankau, R., Imbriani, J. L., & Bell, A. H. (1976). SEM observations on nematode cuticle penetration by
Bacillus penetrans. Journal of nematology, 8(2), 179.
 Kokalis-Burelle, N. (2015). Pasteuria penetrans for control of Meloidogyne incognita on tomato and
cucumber, and M. arenaria on snapdragon. Journal of nematology, 47(3), 207.
 Terefe, M., Tefera, T., & Sakhuja, P. K. (2012). Biocontrol (Formulation of Bacillus firmus (BioNem)) of
root-knot nematode, Meloidogyne incognita on tomato plants in the field. Ethiopian Journal of
Agricultural Sciences, 22(1), 102-116.
 Chen, J., Moore, W. H., Yuen, G. Y., Kobayashi, D., & Caswell-Chen, E. P. (2006). Influence of
Lysobacter enzymogenes strain C3 on nematodes. Journal of nematology, 38(2), 233.
 Sources

 Thanks !
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