SHORT COMMUNICATION

Identification and nematicidal activity of bacteria isolatedfrom 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 wereisolated from cow dung. Among these, 59 isolates displayednematicidal activity against the model nematodeCaenorhabditis elegans. Of the 59 bacterial strains, 17killed >90 % of the tested nematodes within 1 h. Based ontheir 16S rRNA sequences, these 17 strains were identifiedas Alcaligenes faecalis, Bacillus cereus, Proteus penneri,Providencia rettgeri, Pseudomonas aeruginosa, Pseudomo-nas otitidis, Staphylococcus sciuri, Staphylococcus xylosus,Microbacterium aerolatum, Pseudomonas beteli. Amongthese 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 aplant pathogenic nematode Meloidogyne incognita. This isthe first report demonstrating nematicidal activity for strainsin genera Proteus, Providencia and Staphylococcus.

Keywords Bacteria . Cow dung . Nematicidal activity . 16SrRNA . Volatile organic compound

Plant-parasitic nematodes are important pathogens of cropsand trees. They have been reported to cause damage worthover US $ 150 billion per year worldwide (Abad et al.2008). Several strategies have been deployed for the controlof nematodes. These strategies include cultural practicessuch as crop rotation, developing and planting resistant cropvarieties, and applying chemical nematicides or biologicalnematicidal agents. Chemical nematicides are usually moreeffective than other strategies, but have caused significantenvironmental problems due to their toxic side effects on theenvironment. So, biological control of nematodes has

attracted increasing scientific interest due to its minimalenvironmental side effects.

Recently, microorganisms have attracted a lot of attentionas potential nematode bio-control agents due to their nem-aticidal activities and environmental friendliness. The pastfew years have seen the development of bacterial secondarymetabolites as an important source of new biologicallyactive compounds for a variety of purposes (Lin et al.2005; Piel 2009; Rahman et al. 2010). Many bacteria canproduce biologically active volatile organic compounds(VOCs). For example, some mycobacteria were proved toemit VOCs (McNerney et al. 2012). Pseudomonas canproduce small chain alcohols, ketones, diacetyl, and esters,which can all act as natural chemoattractants for nematode(Bargmann et al. 1993). Seven bacterial species includingBacillus simplex, B. subtilis, B. weihenstephanensis,Microbacterium oxydans, Stenotrophomonas maltophilia,Streptomyces lateritius and Serratia marcescens could pro-duce nematicidal VOCs (Gu et al. 2007). Bacillusmegaterium produced VOCs that could inhibit nematodeegg hatching and reduce infection by the nematodeMeloidogyne incognita (Huang et al. 2010). A new speciesLysinibacillus mangiferahumi isolated from rhizosphere soilalso showed nematicidal activity against M. incognitathrough its VOCs (Yang et al. 2012).

Our previous work evaluated the nematicidal activity ofendophytic bacteria from plants (Zheng et al. 2008). In thepresent work, the nematicidal activity of bacteria isolatedfrom cow dung was investigated and the production ofVOCs was also examined.

The bacterial strains were isolated from a cow dungsample collected in Kunming, China. The dung sample(10 g) was suspended into 100 mL sterile water. Afterfiltration, the suspension was diluted to 10−4–10−6. Thediluted samples were spread onto LB (Luria-Bertani:tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0) andNB (nutrient broth: peptone 10 g, beef extract 5 g, NaCl 5 g,

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 ofEducation, Yunnan University, Kunming 650091 Yunnan, Chinae-mail: ghli1223@hotmail.com

Ann Microbiol (2014) 64:407–411DOI 10.1007/s13213-013-0660-7

pH 7.0–7.2) plates. After incubation for 2 days at 30 °C,bacterial colonies were picked randomly and cultured on LBplates. Each bacterial colony was streaked three times con-tinuously and picked again to insure pure culture. Twohundred and nineteen strains were obtained and depositedin the Key Laboratory for Conservation and Utilization ofBio-resources of Yunnan Province, China. For long-termstorage, bacteria were maintained at −80 °C in LB mediumcontaining 20 % glycerol. For subsequent experiments,stock cultures were streaked onto LB plates and incubatedat 28 °C for 24 h. Individual isolates were grown in flaskscontaining 50 mL LB. Flasks were incubated for 4 days at30 °C at 180 rpm, and the cultures were centrifuged, the cellmass discarded, and the liquid broth was retained for sub-sequent experiments.

Caenorhabditis elegans (Maupas) Dougherty was cul-tured on oatmeal medium (20 g oatmeal and 80 mL water)at 25 °C for 7 days, and then refrigerated prior to use. Aplant-parasitic nematode Meloidogyne incognita (Kotoidand White) was also used. Both organisms were collectedfrom greenhouse-grown tomato plants using a protocol de-scribed previously (Hussey and Barker 1973). Nematodeeggs were treated in 0.5 % NaOCl and were rinsed carefullywith water. Second-stage juveniles (J2) were collected byhatching eggs on sieves with 10 μm openings suspendedover deionized water in a 3 cm plate.

Nematicidal activity of bacteria towards C. elegans wastested according to Yang et al. (2010) with some modifica-tions. Fermentation broth (1 mL) of the tested strains wasadded to eaaxh compartment of 16-well plates containingapproximately 100 nematodes per well. Each treatment wasreplicated three times while LB medium was used as anegative control. All 16-well plates were incubated at roomtemperature. The nematodes were considered dead whenthey did not move upon physical stimulus with a fineneedle. The numbers of live and inactive nematodes werecounted after different incubation times (1, 3, 6, 24 h).Nematicidal activity (NA) was calculated using the for-mula: NA=IN/SN×100 % ([N: the number of immobilenematodes; SN: the sum of all nematodes counted (SN>100)]. The NA≥90 %, 50 %≤NA<90 %, 20 %≤NA<50 % or NA<20 % at 24 h were considered to havestrong, moderate, relatively low or no NA, respectively.Data were analyzed using the analysis of variance(ANOVA), and means were compared by the test of leastsignificant difference (LSD) at P=0.05 using SPSS 13.0for Windows (SPSS, Chicago, IL).

Nematicidal activity of bacterial VOCs was testedaccording to the method described by Gu et al. (2007).Briefly, 5 mL fresh bacterial culture was added into onecompartment of a three-compartmented Petri dish and alayer of water agar (WA) medium was added to the othertwo compartments. For the two compartments with WA,

approximate 100 nematodes of C. elegans and M. incognitawere added respectively. Plates were immediately wrappedwith parafilm to prevent the escape of the volatiles. Afterincubating at room temperature for 24 h, the numbers ofmobile and immobile nematodes were recorded by countingunder a microscope. The same volume of LB was used ascontrol. For each bacterium, the test was carried out intriplicate and the average calculated. Data were analyzedusing the same method described above.

Bacterial strains were cultured overnight in 5 mL LBmedium at 37 °C and then centrifuged at 8,000 rpm for2 min. First, the cell pellet was washed and re-suspended in0.5 mL TE-buffer (10 mM Tris–HCl and 1 mM EDTA,pH 8.0). Next, 250 μL phenol and 250 μL chloroform wasmixed gently by inversion and was centrifuged at 4 °C for15 min. After phase separation, the aqueous phase wasprecipitated using isopropanol. The pellet was suspendedin 20–50 μL ddH2O. Finally, PCR amplification wasperformed in a total reaction volume of 50 μL comprising37.75 μL sterile distilled water, 5 μL 10×exTaq buffer, 4 μLof each dNTP (2.5 mM), 1 μL of each primer (10 μM),0.25 μL exTaq DNA polymerase (code DRR001A, TaKaRaBio, Otsu, Japan), and 1 μL template DNA (20 ng). Ampli-fication was performed under the following conditions: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and72 °C for 2 min; and an additional extension step at 72 °Cfor 10 min. Amplification was performed using the univer-sal 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 resultingsequences were analyzed with the BLAST algorithm of theRibosomal Database Project II (RDP II) (Maidak et al.2001). The 16S rRNA gene sequences have been depositedin the NCBI nucleotide sequence database. Phylogenetictrees were constructed by the neighbor-joining method(Saitou and Nei 1987) using the program MEGA (version5.0), in which Wautersiella falsenii (AM084341) was usedas outgroup. The robustness at the individual branchingpoints was estimated by bootstrapping with 1,000 replica-tions (Felsenstein 1985).

A total of 219 bacterial strains were assayed for theirnematicidal activity against C. elegans. Fifty-nine isolates(accounting to 26.94 %) displayed more than 20 % NAagainst C. elegans at 24 h. Among them, 17 had very highNA (NA≥90 %) at 1 h. These 17 strains were chosen forfurther experiments. Thirty strains (13.70 % of the total) and4 (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 NAat 4.19 % at 24 h. For active isolate, the activity wassignificantly more than the control (P<0.01).

Seventeen strains with ≥90 % NA in 1 h in the aboveexperiment were further tested for their nematicidal activity

408 Ann Microbiol (2014) 64:407–411

through VOCs by the three compartmented Petri dish meth-od. In the sealed dish filled with nematicidal VOCs, nema-todes gradually reduced their movement within 1–12 h of

incubation, and mostly stopped action. When these immo-bile nematodes were transferred to tap water, they could notbe revived. The results showed that 14 strains could produceVOCs to kill nematodes (Table 1). Among them, 7 and 10strains could kill 100 % C. elegans andM. incognita at 24 h,respectively.

Seventeen strains with high NA (1 h, NA ≥90 %) wereidentified based on their 16S rRNA gene. The nucleotidesequence data reported in this paper have been deposited inGenBank. The identification results are presented in Table 2.Their phylogenetic relationships based on the 16S rRNAgene are shown in Fig. 1. Among these strains, culturefiltrates of Pseudomonas aeruginosa have been reported tokill the root-knot nematode Meloidogyne javanica in vitro(Ali et al. 2002). Alcaligenes faecalis showed antagonisticactivity againstM. incogita (Zhou 2005) and Bacillus cereusinhibited M. javanica (Oka et al. 1993). However, strains ofProteus, Providencia and Staphylococcus have never beenreported to have nematicidal activity.

Naturally occurring volatile compounds are of signif-icant importance in agriculture and forestry. Chemicalfumigants can lead to the eradication of beneficial or-ganisms, and lead to a negative shift in the biologicalequilibrium (Gamliel et al. 2000). Thus, non-chemicalmethods that can effectively control plant diseases arehighly desirable. Our further experiment proved that 14isolates with high nematicidal activity could produceVOCs against M. incognita and C. elegans. An earlierstudy showed that strains of Staphylococcus sp. andBacillus sp. as could kill fungi by producing volatilecompounds (Zou et al. 2007). However, our experiment

Table 1 Nematicidal activity of volatile organic compounds (VOCs)from 17 bacteria against Caenorhabditis elegans and Meloidogyneincognita Data are given as mean ± standard deviation

Isolate Mortality (%) after exposure to bacterial VOCs

C. elegans M. incognita

12 h 24 h 12 h 24 h

Control(broth)

1.60±0.34 3.37±1.35 2.54±2.14 8.89±1.52

CD232 26.33±2.16 100.00±0.00 15.36±2.91 100.00±0.00

CD243 20.35±2.83 100.00±0.00 24.51±5.49 100.00±0.00

CD204 21.73±1.12 99.34±3.92 25.65±4.29 97.34±7.56

CD129 22.82±3.66 100.00±0.00 18.12±0.54 98.45±8.05

CD212 24.50±4.54 98.32±7.50 24.80±0.87 100.00±0.00

CD256 29.94±3.96 100.00±0.00 21.33±5.27 100.00±0.00

CD261 21.11±2.95 99.64±3.45 17.39±4.08 99.33±5.87

CD97 23.21±0.63 99.69±3.19 24.44±0.91 100.00±0.00

CD250 16.92±4.75 100.00±0.00 17.49±4.43 100.00±0.00

CD260 18.41±1.53 97.34±5.28 23.22±4.18 100.00±0.00

CD257 22.04±5.54 91.44±0.74 22.54±2.94 94.01±5.62

CD9 22.31±2.99 100.00±0.00 28.40±5.11 100.00±0.00

CD98 22.50±3.17 98.93±4.38 17.33±5.50 100.00±0.00

CD142 47.40±1.41 100.00±0.00 41.83±7.39 100.00±0.00

CD245 5.26±3.99 9.24±1.09 2.69±1.93 11.23±3.48

CD205 3.81±1.91 8.14±2.74 2.01±0.05 14.78±1.08

CD237 4.13±1.37 8.35±0.90 3.40±0.00 13.61±5.92

Table 2 Similarity of 16SrDNA sequences of bacteriawith the closely related ones inthe Ribosomal DatabaseProject II

Isolate Accession no. Closest RDP II strain and accession no. Similarity score

CD232 JX871325 Alcaligenes faecalis EU921230 0.999

CD243 JX871327 Alcaligenes faecalis HQ848384 0.999

CD204 JX871322 Pseudomonas beteli JN216878 1.000

CD129 JX871320 Proteus penneri AB82277 0.998

CD212 JX871324 Proteus penneri AB682277 0.996

CD256 JX871330 Providencia rettgeri HQ009881 0.997

CD261 JX871333 Alcaligenes faecalis HQ202537 0.999

CD97 JX871318 Staphylococcus sciuri EU095646 0.995

CD250 JX871329 Staphylococcus xylosus D83374 0.997

CD260 JX871332 Bacillus cereus AJ577283 1.000

CD257 JX871331 Alcaligenes faecalis GQ284565 0.999

CD9 JX871317 Bacillus cereus AF290547 1.000

CD98 JX871319 Bacillus cereus AF290555 1.000

CD142 JX871321 Microbacterium aerolatum AJ309929 0.997

CD245 JX871328 Pseudomonas aeruginosa FJ665501 1.000

CD205 JX871323 Alcaligenes faecalis HQ72766 0.999

CD237 JX871326 Pseudomonas otitidis EU301769 1.000

Ann Microbiol (2014) 64:407–411 409

is the first to demonstrate that strains of Alcaligenesfaecalis, Pseudomonas beteli, Pseudomonas otitidis,Proteus penneri, Providencia rettgeri, Staphylococcussciuri and Staphylococcus xylosus could produce vola-tile nematicidal components. These isolates may havethe potential to be developed as effective biocontrolagents against nematodes.

As one of the enormous sources of new medicinaland agricultural products, bacteria represent a huge po-tential of new natural nematicides. To our knowledge,this is the first study demonstrating bacteria with nem-aticidal activity isolated from cow dung. The results ofthe present work clearly confirmed that bacteria isolatedfrom unusual environments can be an abundant sourceof novel nematicidal agents.

Acknowledgments The work was supported by grants from the“973” Program of China (2013CB127505, 2012CB722208) and theNSFC (30960007), the Young Academic and Technical Leader RaisingFoundation of Yunnan Province (2010CI023), and the Program fromYunnan Provincial Company (2010yn17) and the China National To-bacco Corporation (110201002023).

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CD232 (JX871325)

CD257 (JX871331)

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