Ann Microbiol (2014) 64:407411

DOI 10.1007/s13213-013-0660-7

SHORT COMMUNICATION

Identification and nematicidal activity of bacteria isolated

from cow dung
Hao Lu & Xin Wang & Keqin Zhang & Youyao Xu & Liang Zhou &
Guohong Li

Received: 28 September 2012 / Accepted: 2 May 2013 / Published online: 18 May 2013
# Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract Two hundred and nineteen bacterial strains were

isolated from cow dung. Among these, 59 isolates displayed
nematicidal activity against the model nematode
Caenorhabditis elegans. Of the 59 bacterial strains, 17
killed >90 % of the tested nematodes within 1 h. Based on
their 16S rRNA sequences, these 17 strains were identified
as Alcaligenes faecalis, Bacillus cereus, Proteus penneri,
Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas otitidis, Staphylococcus sciuri, Staphylococcus xylosus,
Microbacterium aerolatum, Pseudomonas beteli. Among
these 17 strains, 14 produced volatile organic compound(s)
that inhibited the mobility of the C. elegans nematodes.
These 14 strains also showed nematicidal activity against a
plant pathogenic nematode Meloidogyne incognita. This is
the first report demonstrating nematicidal activity for strains
in genera Proteus, Providencia and Staphylococcus.
Keywords Bacteria . Cow dung . Nematicidal activity . 16S
rRNA . Volatile organic compound
Plant-parasitic nematodes are important pathogens of crops
and trees. They have been reported to cause damage worth
over US $ 150 billion per year worldwide (Abad et al.
2008). Several strategies have been deployed for the control
of nematodes. These strategies include cultural practices
such as crop rotation, developing and planting resistant crop
varieties, and applying chemical nematicides or biological
nematicidal agents. Chemical nematicides are usually more
effective than other strategies, but have caused significant
environmental problems due to their toxic side effects on the
environment. So, biological control of nematodes has
H. Lu : X. Wang : K. Zhang : Y. Xu : L. Zhou : G. Li (*)
Key Laboratory for Conservation and Utilization of Bio-resource,
and Key Laboratory for Microbial Resources of the Ministry of
Education, Yunnan University, Kunming 650091 Yunnan, China
e-mail: ghli1223@hotmail.com

attracted increasing scientific interest due to its minimal

environmental side effects.
Recently, microorganisms have attracted a lot of attention
as potential nematode bio-control agents due to their nematicidal activities and environmental friendliness. The past
few years have seen the development of bacterial secondary
metabolites as an important source of new biologically
active compounds for a variety of purposes (Lin et al.
2005; Piel 2009; Rahman et al. 2010). Many bacteria can
produce biologically active volatile organic compounds
(VOCs). For example, some mycobacteria were proved to
emit VOCs (McNerney et al. 2012). Pseudomonas can
produce small chain alcohols, ketones, diacetyl, and esters,
which can all act as natural chemoattractants for nematode
(Bargmann et al. 1993). Seven bacterial species including
Bacillus simplex, B. subtilis, B. weihenstephanensis,
Microbacterium oxydans, Stenotrophomonas maltophilia,
Streptomyces lateritius and Serratia marcescens could produce nematicidal VOCs (Gu et al. 2007). Bacillus
megaterium produced VOCs that could inhibit nematode
egg hatching and reduce infection by the nematode
Meloidogyne incognita (Huang et al. 2010). A new species
Lysinibacillus mangiferahumi isolated from rhizosphere soil
also showed nematicidal activity against M. incognita
through its VOCs (Yang et al. 2012).
Our previous work evaluated the nematicidal activity of
endophytic bacteria from plants (Zheng et al. 2008). In the
present work, the nematicidal activity of bacteria isolated
from cow dung was investigated and the production of
VOCs was also examined.
The bacterial strains were isolated from a cow dung
sample collected in Kunming, China. The dung sample
(10 g) was suspended into 100 mL sterile water. After
filtration, the suspension was diluted to 104106. The
diluted samples were spread onto LB (Luria-Bertani:
tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0) and
NB (nutrient broth: peptone 10 g, beef extract 5 g, NaCl 5 g,

408

pH 7.07.2) plates. After incubation for 2 days at 30 C,

bacterial colonies were picked randomly and cultured on LB
plates. Each bacterial colony was streaked three times continuously and picked again to insure pure culture. Two
hundred and nineteen strains were obtained and deposited
in the Key Laboratory for Conservation and Utilization of
Bio-resources of Yunnan Province, China. For long-term
storage, bacteria were maintained at 80 C in LB medium
containing 20 % glycerol. For subsequent experiments,
stock cultures were streaked onto LB plates and incubated
at 28 C for 24 h. Individual isolates were grown in flasks
containing 50 mL LB. Flasks were incubated for 4 days at
30 C at 180 rpm, and the cultures were centrifuged, the cell
mass discarded, and the liquid broth was retained for subsequent experiments.
Caenorhabditis elegans (Maupas) Dougherty was cultured on oatmeal medium (20 g oatmeal and 80 mL water)
at 25 C for 7 days, and then refrigerated prior to use. A
plant-parasitic nematode Meloidogyne incognita (Kotoid
and White) was also used. Both organisms were collected
from greenhouse-grown tomato plants using a protocol described previously (Hussey and Barker 1973). Nematode
eggs were treated in 0.5 % NaOCl and were rinsed carefully
with water. Second-stage juveniles (J2) were collected by
hatching eggs on sieves with 10 m openings suspended
over deionized water in a 3 cm plate.
Nematicidal activity of bacteria towards C. elegans was
tested according to Yang et al. (2010) with some modifications. Fermentation broth (1 mL) of the tested strains was
added to eaaxh compartment of 16-well plates containing
approximately 100 nematodes per well. Each treatment was
replicated three times while LB medium was used as a
negative control. All 16-well plates were incubated at room
temperature. The nematodes were considered dead when
they did not move upon physical stimulus with a fine
needle. The numbers of live and inactive nematodes were
counted after different incubation times (1, 3, 6, 24 h).
Nematicidal activity (NA) was calculated using the formula: NA=IN/SN100 % ([N: the number of immobile
nematodes; SN: the sum of all nematodes counted (SN>
100)]. The NA90 %, 50 %NA<90 %, 20 %NA<
50 % or NA<20 % at 24 h were considered to have
strong, moderate, relatively low or no NA, respectively.
Data were analyzed using the analysis of variance
(ANOVA), and means were compared by the test of least
significant difference (LSD) at P=0.05 using SPSS 13.0
for Windows (SPSS, Chicago, IL).
Nematicidal activity of bacterial VOCs was tested
according to the method described by Gu et al. (2007).
Briefly, 5 mL fresh bacterial culture was added into one
compartment of a three-compartmented Petri dish and a
layer of water agar (WA) medium was added to the other
two compartments. For the two compartments with WA,

Ann Microbiol (2014) 64:407411

approximate 100 nematodes of C. elegans and M. incognita

were added respectively. Plates were immediately wrapped
with parafilm to prevent the escape of the volatiles. After
incubating at room temperature for 24 h, the numbers of
mobile and immobile nematodes were recorded by counting
under a microscope. The same volume of LB was used as
control. For each bacterium, the test was carried out in
triplicate and the average calculated. Data were analyzed
using the same method described above.
Bacterial strains were cultured overnight in 5 mL LB
medium at 37 C and then centrifuged at 8,000 rpm for
2 min. First, the cell pellet was washed and re-suspended in
0.5 mL TE-buffer (10 mM TrisHCl and 1 mM EDTA,
pH 8.0). Next, 250 L phenol and 250 L chloroform was
mixed gently by inversion and was centrifuged at 4 C for
15 min. After phase separation, the aqueous phase was
precipitated using isopropanol. The pellet was suspended
in 2050 L ddH 2O. Finally, PCR amplification was
performed in a total reaction volume of 50 L comprising
37.75 L sterile distilled water, 5 L 10exTaq buffer, 4 L
of each dNTP (2.5 mM), 1 L of each primer (10 M),
0.25 L exTaq DNA polymerase (code DRR001A, TaKaRa
Bio, Otsu, Japan), and 1 L template DNA (20 ng). Amplification was performed under the following conditions: 94
C for 5 min; 30 cycles of 94 C for 30 s, 55 C for 30 s, and
72 C for 2 min; and an additional extension step at 72 C
for 10 min. Amplification was performed using the universal bacterial primers 20F and 1500R (Weisburg et al. 1991)
or 27F and 1492R (DeLong 1992). To identify the isolates,
amplicons were sequenced by BGI, China, and the resulting
sequences were analyzed with the BLAST algorithm of the
Ribosomal Database Project II (RDP II) (Maidak et al.
2001). The 16S rRNA gene sequences have been deposited
in the NCBI nucleotide sequence database. Phylogenetic
trees were constructed by the neighbor-joining method
(Saitou and Nei 1987) using the program MEGA (version
5.0), in which Wautersiella falsenii (AM084341) was used
as outgroup. The robustness at the individual branching
points was estimated by bootstrapping with 1,000 replications (Felsenstein 1985).
A total of 219 bacterial strains were assayed for their
nematicidal activity against C. elegans. Fifty-nine isolates
(accounting to 26.94 %) displayed more than 20 % NA
against C. elegans at 24 h. Among them, 17 had very high
NA (NA90 %) at 1 h. These 17 strains were chosen for
further experiments. Thirty strains (13.70 % of the total) and
4 (1.83 % of the total) exhibited moderate (50 %NA<
90 %) and low NA (20 %NA<50 %) at 24 h, respectively.
At the same time, the control of LB medium showed the NA
at 4.19 % at 24 h. For active isolate, the activity was
significantly more than the control (P<0.01).
Seventeen strains with 90 % NA in 1 h in the above
experiment were further tested for their nematicidal activity

Ann Microbiol (2014) 64:407411

409

Table 1 Nematicidal activity of volatile organic compounds (VOCs)

from 17 bacteria against Caenorhabditis elegans and Meloidogyne
incognita Data are given as mean standard deviation
Isolate

Mortality (%) after exposure to bacterial VOCs

C. elegans
12 h

M. incognita
24 h

12 h

24 h

Control
(broth)
CD232
CD243
CD204
CD129
CD212
CD256
CD261
CD97
CD250
CD260
CD257
CD9

1.600.34

3.371.35

2.542.14

8.891.52

26.332.16
20.352.83
21.731.12
22.823.66
24.504.54
29.943.96
21.112.95
23.210.63
16.924.75
18.411.53
22.045.54
22.312.99

100.000.00
100.000.00
99.343.92
100.000.00
98.327.50
100.000.00
99.643.45
99.693.19
100.000.00
97.345.28
91.440.74
100.000.00

15.362.91
24.515.49
25.654.29
18.120.54
24.800.87
21.335.27
17.394.08
24.440.91
17.494.43
23.224.18
22.542.94
28.405.11

100.000.00
100.000.00
97.347.56
98.458.05
100.000.00
100.000.00
99.335.87
100.000.00
100.000.00
100.000.00
94.015.62
100.000.00

CD98
CD142
CD245
CD205
CD237

22.503.17
47.401.41
5.263.99
3.811.91
4.131.37

98.934.38
100.000.00
9.241.09
8.142.74
8.350.90

17.335.50
41.837.39
2.691.93
2.010.05
3.400.00

100.000.00
100.000.00
11.233.48
14.781.08
13.615.92

through VOCs by the three compartmented Petri dish method. In the sealed dish filled with nematicidal VOCs, nematodes gradually reduced their movement within 112 h of

Table 2 Similarity of 16S

rDNA sequences of bacteria
with the closely related ones in
the Ribosomal Database
Project II

incubation, and mostly stopped action. When these immobile nematodes were transferred to tap water, they could not
be revived. The results showed that 14 strains could produce
VOCs to kill nematodes (Table 1). Among them, 7 and 10
strains could kill 100 % C. elegans and M. incognita at 24 h,
respectively.
Seventeen strains with high NA (1 h, NA 90 %) were
identified based on their 16S rRNA gene. The nucleotide
sequence data reported in this paper have been deposited in
GenBank. The identification results are presented in Table 2.
Their phylogenetic relationships based on the 16S rRNA
gene are shown in Fig. 1. Among these strains, culture
filtrates of Pseudomonas aeruginosa have been reported to
kill the root-knot nematode Meloidogyne javanica in vitro
(Ali et al. 2002). Alcaligenes faecalis showed antagonistic
activity against M. incogita (Zhou 2005) and Bacillus cereus
inhibited M. javanica (Oka et al. 1993). However, strains of
Proteus, Providencia and Staphylococcus have never been
reported to have nematicidal activity.
Naturally occurring volatile compounds are of significant importance in agriculture and forestry. Chemical
fumigants can lead to the eradication of beneficial organisms, and lead to a negative shift in the biological
equilibrium (Gamliel et al. 2000). Thus, non-chemical
methods that can effectively control plant diseases are
highly desirable. Our further experiment proved that 14
isolates with high nematicidal activity could produce
VOCs against M. incognita and C. elegans. An earlier
study showed that strains of Staphylococcus sp. and
Bacillus sp. as could kill fungi by producing volatile
compounds (Zou et al. 2007). However, our experiment

Isolate

Accession no.

Closest RDP II strain and accession no.

Similarity score

CD232
CD243
CD204
CD129

JX871325
JX871327
JX871322
JX871320

Alcaligenes faecalis
Alcaligenes faecalis
Pseudomonas beteli
Proteus penneri

EU921230
HQ848384
JN216878
AB82277

0.999
0.999
1.000
0.998

CD212
CD256
CD261
CD97
CD250
CD260
CD257
CD9
CD98
CD142
CD245
CD205
CD237

JX871324
JX871330
JX871333
JX871318
JX871329
JX871332
JX871331
JX871317
JX871319
JX871321
JX871328
JX871323
JX871326

Proteus penneri
Providencia rettgeri
Alcaligenes faecalis
Staphylococcus sciuri
Staphylococcus xylosus
Bacillus cereus
Alcaligenes faecalis
Bacillus cereus
Bacillus cereus
Microbacterium aerolatum
Pseudomonas aeruginosa
Alcaligenes faecalis
Pseudomonas otitidis

AB682277
HQ009881
HQ202537
EU095646
D83374
AJ577283
GQ284565
AF290547
AF290555
AJ309929
FJ665501
HQ72766
EU301769

0.996
0.997
0.999
0.995
0.997
1.000
0.999
1.000
1.000
0.997
1.000
0.999
1.000

410

Ann Microbiol (2014) 64:407411

CD232 (JX871325)
CD257 (JX871331)
98

99

CD205 (JX871323)
CD243 (JX871327)
CD261 (JX871333)
Alcaligenes faecalis strain GT (AJ242986)

23

CD256 (JX871330)

97

Providencia rettgeri DSM 4542T (AM040492)

Proteus penneri NCTC 12737T (DQ885258)

94

CD129 (JX871320)

99
90
53

CD212 (JX871324)
CD204 (JX871322)
Pseudomonas beteli

99

ATCC 19861T (AB021406)

Pseudomonas otitidis DSM 17224T (AY953147)

92
97

CD237 (JX871326)
CD245 (JX871328)
100

CD142 (JX871321)
Microbacterium aerolatum

DSM 14217T (AJ309929)

CD98 (JX871319)
67

Bacillus cereus ATCC 14579T (AE016877)

99

CD9 (JX871317)
CD260 (JX871332)
98 84

CD97 (JX871318)
Staphylococcus sciuri

98
99

DSM 20345T (AJ421446)

CD250 (JX871329)
Staphylococcus xylosus ATCC 29971T (D83374)
Wautersiella falsenii

NF993T (AM084341)

0.1

Fig. 1 Phylogenetic relationships among strains isolated in this study

and those closely related in DRP II. The tree was obtained by the
neighbor-joining method of analyses based on the 16S rRNA gene
sequences. Wautersiella falsenii (AM084341) was used as an

outgroup. Numbers at nodes indicate bootstrap percentages derived

from 1,000 replications. GenBank accession numbers for each strain
are indicated in parentheses adjacent to each strain name. Bar 0.1
nucleotide substitutions per 100 nt

is the first to demonstrate that strains of Alcaligenes

faecalis, Pseudomonas beteli, Pseudomonas otitidis,
Proteus penneri, Providencia rettgeri, Staphylococcus
sciuri and Staphylococcus xylosus could produce volatile nematicidal components. These isolates may have
the potential to be developed as effective biocontrol
agents against nematodes.
As one of the enormous sources of new medicinal
and agricultural products, bacteria represent a huge potential of new natural nematicides. To our knowledge,
this is the first study demonstrating bacteria with nematicidal activity isolated from cow dung. The results of
the present work clearly confirmed that bacteria isolated
from unusual environments can be an abundant source
of novel nematicidal agents.

Acknowledgments The work was supported by grants from the

973 Program of China (2013CB127505, 2012CB722208) and the
NSFC (30960007), the Young Academic and Technical Leader Raising
Foundation of Yunnan Province (2010CI023), and the Program from
Yunnan Provincial Company (2010yn17) and the China National Tobacco Corporation (110201002023).

References
Abad P, Gouzy J, Aury JM et al (2008) Genome sequence of the
metazoan plant-parasitic nematode Meloidogyne incognita. Nat
Biotechnol 26(8):909915
Ali NI, Siddiqui IA, Shaukat S, Zaki MJ (2002) Nematicidal activity of
some strains of Pseudomonas spp. Soil Biol Biochem 34:1051
1058

Ann Microbiol (2014) 64:407411

Bargmann CI, Hartwieg E, Horvitz HR (1993) Odorant-selective genes
and neurons mediate olfaction in C. elegans. Cell 74:515527
DeLong EF (1992) Archaea in coastal marine environments. Proc Natl
Acad Sci USA 89:56855689
Felsenstein J (1985) Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39:783791
Gamliel A, Austerweil M, Kritzman G (2000) Non-chemical approach
to soilborne pest management-organic amendments. Crop Prot
9:847853
Gu YQ, Zhou JP, Zou CS, Mo MH, Zhang KQ (2007) Evaluation and
identification of potential organic nematocidal volatiles from soil
bacteria. Soil Biol Biochem 39:25672575
Huang Y, Xu CK, Ma L, Zhang KQ, Duan CQ, Mo MH (2010)
Characterisation of volatiles produced from Bacillus megaterium
YFM3.25 and their nematicidal activity against Meloidogyne
incognita. Eur J Plant Pathol 126:417422
Hussey RS, Barker KR (1973) A comparison of methods of collecting
inocula of Meloidogyne spp., including a new technique. Plant
Dis Rep 57:10251028
Lin J, Yan XJ, Zheng L, Ma HH, Chen HM (2005) Cytotoxicity and
apoptosis induction of some selected marine bacteria metabolites.
J Appl Microbiol 99:13731382
Maidak BL, Cole JR, Lilburn TG, Parker CT Jr, Saxman PR,
Stredwick JM, Garrity GM, Li B, Olsen GJ, Pramanik S, Schmidt
TM, Tiedje JM (2001) The RDP-II (Ribosomal database project).
Nucleic Acids Res 28:173174
McNerney R, Mallard K, Okolo PI, Turner C (2012) Production of
volatile organic compounds by mycobacteria. FEMS Microbiol
Lett 328:150156

411
Oka Y, Chet I, Spiegel Y (1993) Control of the rootknot nematode
meloidogyne javanica by Bacillus cereus. Biocontrol Sci Technol
3:115126
Piel J (2009) Metabolites from symbiotic bacteria. Nat Prod Rep
26:338362
Rahman H, Austin B, Mitchell WJ, Morris PC, Jamieson DJ, Adams
DR, Spragg AM, Schweizer M (2010) Novel anti-infective compounds from marine bacteria. Mar Drugs 8:498518
Saitou N, Nei M (1987) The neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol Biol Evol 4:406425
Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol
173:697703
Yang ZS, Li GH, Zhao PJ, Zheng X, Luo SL, Li L, Niu XM, Zhang
KQ (2010) Nematicidal activity of Trichoderma spp. and isolation of an active compound. World J Microb Biotechnol
26:22972302
Yang LL, Huang Y, Liu J, Ma L, Mo MH, Li WJ, Yang FX (2012)
Lysinibacillus mangiferahumi sp. nov., a new bacterium producing nematicidal volatiles. Antonie van Leeuwenhoek 102:5359
Zheng LJ, Li GH, Wang XB, Pan WZ, Li L, Lv H, Liu FF, Dang LZ,
Mo MH, Zhang KQ (2008) Nematicidal endophytic bacteria
obtained from plants. Ann Microbiol 58(4):569572
Zhou LJ (2005) Application of GFP on the study of Meloidogyne
incogita antagonistic Alcaligenes faecalis. Dissertation, Fujian
Agriculture and Forestry University, China
Zou CS, Mo MH, Gu YQ, Zhou JP, Zhang KQ (2007) Possible
contributions of volatile-producing bacteria to soil fungistasis.
Soil Biol Biochem 39:23712379