Transcript Hazem Mohamed Elewa Abdelnabby_Poster_04

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EXPOSURE OF SOIL-DWELLING NEMATODES TO DIACETYLPHLOROGLUCINOL (DAPG)
Meyer, Susan L.F.1, J.M. Halbrendt2, L.K. Carta1, A.M. Skantar1, T. Liu3, H.M.E. Abdelnabby4, and B.T. Vinyard5.
1USDA, ARS Nematology Laboratory, Beltsville, MD, USA, 2Pennsylvania State University, Biglerville, PA, USA, 3Beijing Academy of Agricultural
and Forestry Science, Beijing, China, 4Benha University, Qaliubia, Egypt, 5USDA, ARS Biometrical Consulting Service, Beltsville, MD, USA.
MATERIALS AND METHODS
Nematode Culture and Collection.
Nematodes were maintained on greenhouse plants (H.
glycines, M. incognita), in corn root explant cultures
(P. scribneri ), or in media with or without Escherichia coli (C.
elegans, P. pacificus and R. rainai). Nematodes were collected
on mesh or from Baermann funnels and surface-rinsed.
RESULTS
Fig. 1. Viability of Meloidogyne incognita eggs and J2 in DAPG
Fig. 1. Meloidogyne incognita eggs and J2 in:
diluted methanol, DAPG in diluted methanol,
or DAPG in water only.
Fig. 2 Viability of Caenorhabditis elegans eggs in DAPG
Fig. 2. Caenorhabditis elegans eggs in DAPG.
130
120
120
100
110
Eggs, DAPG in Water
100
Eggs in Methanol
90
J2, DAPG in Water
Eggs, DAPG in Methanol
J2, DAPG in Methanol
80
80
60
40
6 hr
20
Table 1. Nematode species, life stage assayed,
number per well, and incubation times in DAPG.
Nematode species Life stage # per
well
Incubation Time
H=Hours, D=Days
3 hr
J2 in Methanol
70
1 hr
0
60
0
20
40
60
80
100
0
120
5
10
15
Fig. 3. Viability of Caenorhabditis elegans J2 in DAPG
Fig.
4. 4.
Viability
of Xiphinema
americanum
in DAPG
Fig.
Xiphinema
americanum
adultsadults
in DAPG.
Fig. 3. Caenorhabditis elegans J1 in DAPG.
100
100
1 hr
80
3 hr
Percentage viable adults
Percentage viable adults
Percentage
ViableJ2
J2
Perc
entage viable
80
60
24 hr
40
20
48 hr
0
60
40
1 day
20
2 days
0
0
5
10
15
DAPG
(µg/mL
water)
DAPGConcentration
Concentration (µg/mL
water)
20
0
20
40
60
Meloidogyne incognita Eggs (Fig. 1): Hatch in water controls was ca. 99.8%. Egg hatch was 91.2%-93.3% in diluted
methanol (0.0045%, 0.045% and 0.45%) without DAPG; methanol alone therefore decreased egg hatch. There was a
significant decrease in egg hatch with DAPG in diluted methanol, with the greatest effect at low DAPG concentrations.
When water alone was used as a solvent for DAPG, there was a significant difference from all other treatments. DAPG
in water was toxic to eggs, resulting in a constant decrease in egg hatch as DAPG concentration increased.
Heterodera
glycines
14
12
19
200
50
1, 3, & 6 H; 1 & 2 D
1, 3, & 6 H; 1 & 2 D
6 H; 1 & 2 D
7D
2D
Meloidogyne
incognita
Eggs
J2
100
50
7D
2D
Meloidogyne incognita J2 (Fig. 1): There was no significant effect of any treatment on J2 viability.
Pratylenchus
scribneri
Pristionchus
pacificus
Juveniles
Females
Eggs
Adults
100
13
58
48
2-3 D
2-4 D
6 H; 1 & 2 D
6 H; 1 & 2 D
Caenorhabditis elegans Eggs (Fig. 2): Egg hatch was stimulated by DAPG at 1 and 3 hours, but no significant effect
was observed at 6 hours, 1 day or 2 days.
Rhabditis
rainai
Eggs
Adults
55
53
6 H; 1 & 2 D
6 H; 1 & 2 D
Xiphinema
americanum
Adults
15
1 &2 D
1 &2 D
Concentrations Tested.
-0, 1, 10, 25, 50, and 75 µg/ml DAPG (all but X. americanum).
-0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 50, and 75 µg/ml
(X. americanum).
-DAPG in methanol at 1, 10 and 100 µg/ml DAPG; 0% (water
only), and 0.0045%, 0.045%, and 0.45% diluted methanol
controls (M. incognita).
There were 4-5 replicate wells per trial, with at least two trials
per treatment (1-2 trials for X. americanum in DAPG > 20 µg/ml).
Statistical Methods.
A 3-factor, nested-effects analysis of covariance model was
conducted using SAS® 9.2 Proc GLIMMIX. Significant
differences (α = 0.05) in the model intercept were identified using
the Extended Shaffer-Royen2 (ESR) multiple comparisons
method, and significant differences (α = 0.05) in model slope
among the nematode species were identified using the
PDMIX8003 SAS macro.
80
DAPG Concentration (µg/mL water)
Eggs
J1
Adults
Eggs
J2
Caenorhabditis
elegans
20
DAPG Concentration (µg/mL water)
DAPG Concentration (µg/mL water)
6 hr
Assays.
Nematodes were incubated in synthetic DAPG (Toronto
Research Chemicals, Inc, North York, Ontario) in microwell
plates at ca. 24 C. Life stages, numbers and incubation times
depended on the life cycle and availability of each species
(Table 1).
48 hr
24 hr
Percentage hatched eggs
Percentage
viable
J2 or
viable egg hatch
or hatched eggs
J2 percentage
viable
Percentage
INTRODUCTION
Some DAPG-producing isolates of Pseudomonas fluorescens
act as biocontrol agents and aid in improving crop yields. DAPG
can affect nematodes, increasing egg hatch of Globodera
rostochiensis but decreasing J2 mobility (Cronin et al., 1997),
and DAPG-producing strains of P. fluorescens can suppress
Meloidogyne javanica numbers (Siddiqui and Shaukat, 2003a,b).
The primary goal of this research was to determine whether
direct application of DAPG to diverse nematode species would
result in either toxic or stimulatory effects. Plant-parasitic
nematodes and free-living nematodes were selected for the
study, to determine whether DAPG would affect target and
nontarget nematodes.
Caenorhabditis elegans J1 (Fig. 3) and Adults: No significant effect of DAPG was observed on J1 or adults.
Xiphinema americanum Adults (Fig. 4): DAPG was toxic to adults of X. americanum. After 24 hours immersion, adult
mobility averaged 97.8% in water controls, dropping rapidly to a mean of 73% at 2 µg/ml DAPG, and 0% in 50 µg/ml
DAPG. This toxic effect was even more pronounced after 2 days.
Heterodera glycines Eggs and J2, Pratylenchus scribneri Juveniles and Females, and Pristionchus pacificus &
Rhabditis ranai Eggs and Adults: No significant effect of DAPG was observed on these nematodes (graphs not shown).
CONCLUSIONS
-Nematode responses to DAPG varied with taxon and life stage. When egg hatch and juvenile/adult viability were
observed, three of the seven tested nematode species responded to the compound. Eggs of five nematode species were
immersed in DAPG; effects on egg hatch varied from stimulatory to inhibitory to no change, depending on the species.
Juveniles of four species were tested; DAPG did not affect juvenile viability of any of those species. Adults from four
species were tested, but DAPG was only toxic to X. americanum adults.
-Methanol is commonly used as a solvent for DAPG, but it was found to alter effects of DAPG on M. incognita eggs.
Consequently, water only was used as a solvent for tests with all other nematode species.
-DAPG production by soil-dwelling bacteria would not directly result in suppression of population numbers of every
plant-pathogenic or bacterial-feeding nematode species. Augmentation of DAPG-producing P. fluorescens populations
for biological control of nematodes should be targeted to pathogenic nematode species that have been shown to be
sensitive to the compound, or used for indirect effects, such as induced systemic resistance.
Acknowledgements: Thanks are extended to Paula Crowley, Sharon Ochs, Maria Hult and Emily Brinker for assistance
in the laboratory and/or with preparation of graphs.
LITERATURE CITED
Cronin, D., Moënne-Loccoz, Y., Fenton, A., Dunne, C., Dowling, D. N. and O’Gara, F. 1997. Role of 2,4-diacetylphloroglucinol in the interactions of the biocontrol pseudomonad strain F113 with the potato cyst nematode Globodera rostochiensis. Appl.
Environ. Micro. 63:1357-1361.
Siddiqui, I. A., and Shaukat, S. S. 2003a. Plant species, host age and host genotype effects on Meloidogyne incognita biocontrol by Pseudomonas fluorescens strain CHA0 and its genetically-modified derivatives. J. Phytopathology 151:231-238.
Siddiqui, I. A., and Shaukat, S. S. 2003b. Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: Importance of bacterial secondary metabolite, 2,4-diacetylphloroglucinol. Soil Biol. Biochem. 35:1615-1623.