)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/bmri/2015/474879.pdf · detected...

Research ArticleEfficacy of Clonostachys rosea and Duddingtonia flagrans inReducing the Haemonchus contortus Infective Larvae

Manoel Eduardo da Silva12 Fabio Ribeiro Braga3

Pedro Mendoza de Gives4 Miguel Angel Mercado Uriostegui4 Manuela Reyes5

Filippe Elias de Freitas Soares6 Lorendane Millena de Carvalho1

Francielle Bosi Rodrigues6 and Jackson Victor de Arauacutejo1

1Departamento de Veterinaria Universidade Federal de Vicosa 36570000 Vicosa MG Brazil2URECOEPAMIG 35650000 Pitangui MG Brazil3Universidade Vila Velha Vila Velha ES Brazil4Area de Helmintologia CENID-Parasitologia INIFAP Jiutepec MOR Mexico5Departamento de Microbiologıa Ambiental y Biotecnologıa UAC CAM Mexico6Programa de Pos Graduacao em Ciencias Farmaceuticas (PPGF-UVV) Universidade Vila Velha 29102920 Vila Velha ES Brazil

Correspondence should be addressed to Fabio Ribeiro Braga fabioribeirobragahotmailcom

Received 29 January 2015 Accepted 10 April 2015

Academic Editor Jose L Campos

Copyright copy 2015 Manoel Eduardo da Silva et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The biocontrol is proven effective in reducing in vitro and in situ free-living stages of major gastrointestinal helminths allowingprogress in reducing losses by parasitism maximizing production and productivity This study aimed at evaluating the predatoryactivity of fungal isolates of Duddingtonia flagrans and Clonostachys rosea species and its association on infective larvae (L

3) of H

contortus in microplots formed by grasses and maintained in a protected environment All groups were added with 10mL of anaqueous suspension with 618 H contortus L

3approximately Group 1 was used as control and only received the infective larvae

Groups 2 and 3 receivedD flagrans chlamydospores and C rosea conidia at doses of 5 times 106 Group 4 received the combination of 5times 106 D flagrans chlamydospores + 5 times 106 C rosea conidia D flagrans and C rosea showed nematicidal effectiveness reducing by915 and 889 respectively the population of H contortus L

3 However when used in combination efficiency decreased to 745

predation of H contortus L3 These results demonstrate the need for further studies to determine the existence of additive effects

synergistic or antagonistic between these species

1 Introduction

Endoparasitic nematodiasis are considered as one of themain factors seriously affecting the livestock industry sincethey cause an important reduction not only in the animalweight but also their main products that is meat andmilk are also diminished [1] Other problems derived fromnematode parasitic infections are the continuous expensesin chemical treatments and eventually the death of younganimals [2] The use of chemical drugs is so far the uniquemethod of anthelmintic control that is established aroundthe world [3] Unfortunately the use of drugs and other

measures of control is in most of the cases not based onthe adequate recommendations that is the general animalstate body weight diagnosis of parasitic burden and otherimportant factors that should be considered to establish asuccessful control as quantity and quality of food selectionof gastrointestinal parasitic genetically resistant animals andthe presence of parasites resistance to chemical drugs [4]

A number of alternatives of control different than the useof chemical drugs are being proposed although the mostpromising measure of control is the use of natural nema-tode antagonists like nematophagous fungi [5 6] Allied tochemical control one of the most promising and sustainable

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 474879 5 pageshttpdxdoiorg1011552015474879

2 BioMed Research International

methods of control is biological control which has showna good effectiveness either in laboratory trials or in pastureexperiments which is an important activity reducing thefree-living stages of mayor gastrointestinal helminths Thismethod can be considered as a useful tool of control whichreduces the economic losses caused by parasites and canpromote a higher animal productivity [7 8]

The species Clonostachys rosea belonging to the orderHypocreales (family Bionectriacea) has being recorded as aparasite of other microfungi and also as a nematode parasiteapart from being a saprophyte fungus [9] This speciescolonizes plants and it has attracted interest by workers as apotential biological control agent since it produces volatileorganic compounds toxic for some living organisms [10 11]

Duddingtonia flagrans is an ldquoold friendrdquo of scientists whoenvision biological control as an alternative to the treatmentof herd animals by chemical means and also in the futureof prevention of high levels of contamination by nematodesin public areas and squares used by humans [3 12] Again itmust be remembered here that the ability to pass through thegastrointestinal tract of domestic animals makes this speciesof fungus an eternal ally in combating nematodes present inthe environment [13]

This fungal species is said to be the ldquomost promisingrdquo Itsmain focus of activity culminates in the predation of infectivehelminth forms (in this case nematode larvae) by means ofsimple adhesive hyphae

The fungus D flagrans belonging to the order Monil-iales (family Moniliaceae) is one of the most promisingmicroorganisms for biological control of ruminant parasiticnematodes [14] This species is harmless to plants animalsand human being [15 16] and it is possible to isolate it mainlyfrom soil samples and animal faeces [17]Thismicroorganismpossesses dual food habits being saprobius in absence ofnematodes and becoming either predatory or parasite ofnematodes in the presence of them [18]

Taking into account the representation of the worldlivestock and its socioeconomics it is important to evaluateimprove and provide the use of technologies that can con-tribute to the development of this segment of agrobusinessThe aim of this study was to evaluate the predatory activity offungal isolates of the speciesC rosea andD flagrans and theirassociation onH contortus infective larvae (L

3) inmicroplots

with graminaceous forage plant

2 Material and Methods

21 Fungi TheC roseaYucatan strain belonging to theCICYcollection was usedThis strain was maintained in 2 water-agar (2WA) at the Department of Helminthology (CENID-PAVET-INIFAP-MEXICO) The D flagrans fungal strain(FTHO-8) belonging to the Fungal Collection of CENID-Parasitologıa Veterinaria INIFAP-Mexico was used Thisstrain was originally obtained from a sheep faecal samplefrom a farm at Fierro del ToroVillage HuitzilacMunicipalityMorelos State Mexico [19]

22 Nematodes A H contortus isolate was originally ob-tained from a naturally infected sheep from ldquoLas Margaritasrdquo

Experimental Sheep Farm (INIFAP) inHueytamalcoMunici-pality in the state of PueblaMexico AH contortus egg donorlambwas orally infectedwith an aqueous suspension contain-ing 350 larvaekg of body weight Twenty days after infectionthe presence of H contortus eggs in faecal samples wasdetected through the McMaster technique Faecal cultureswere prepared by mixing faeces with polystyrene particles inplastic bowls Water was added to the faecal cultures and themixture was homogenized to keep the cultures hydrated andoxygenated to promote better larval hatching [20] Faecal cul-tures were covered with paper foil and incubated for 7 days atroom temperature (25ndash30∘C) Infective larvae were extractedfrom faeces using the Baermann funnel technique [21] Thelarvae were washed several times in density gradients of 50sucrose solution and rinsed and suspended in sterile water

23 Assay The pots were conformed as follows thirty-sixplastic (gardening) pots with a capacity of 100 grams (75times 55mm) were used Seventy grams of soil acquired fromcommercial nurseries was autoclaved at 12 atm and 115∘Cfor 15 minutes and added to the plastic pots Every pot withsoil was added with 006 grams of a commercial mixture ofdifferent generaspecies grass seeds about 70 graminaceousseeds were added to the soil surface in order to obtain ahomogeneous grass growing Six mL of top water was addedto every pot every day during 18 days of the experiment usinga Pasteur pipette to maintain the pots humidity

After germination the pots were randomly divided intofour (4) experimental groups with 8 replicates each Allgroups were added with 10mL average of an acqueos sus-pension with 618 H contortus L

3approximately Group 1 was

used as control and only received the infective larvae Groups2 and 3 received D flagrans chlamydospores and C roseaconidia at doses of 5 times 106 respectively Group 4 received thecombination of 5 times 106 D flagrans chlamydospores + 5 times 106C rosea conidia

The pots were maintained under a shading rate hedgingprotected screen and after 12 days of interaction betweennematophagous fungi and parasitic nematodes the wholenematode population from total soil and grass of every singlepot was recovered using the Baermann Funnel method [21]

24 Location The experiment was conducted in CENID-PAVET-INIFAP-MEXICO located at 18∘5310158400410158401015840N latitudeand 98∘5110158403410158401015840W and 161516 meters above sea level in themunicipality of Jiutepec Morelos Mexico

25 Weather Data Meteorological data including maximumand minimum temperatures relative humidity and rain-fall during the experiment were obtained from the closestexperimental weather station (National Institute for WaterTechnology) Jiutepec Morelos Mexico

26 Statistical Analysis Datawere analysed using anANOVAtest and the Tukey test was used as a complementary tech-nique for discriminating the mean different from the othersData were analysed using the software BioEstat 53

BioMed Research International 3

Table 1 Number and percentage of reduction of Haemonchus contortus L3 recovered by the method of Baermann 12 days after interactionwith fungal isolates Clonostachys rosea and Duddingtonia flagrans and association of these microfungi

Treatment Control C rosea D flagrans D flagrans + C roseaH contortus 118 (plusmn114)a 13 (plusmn19)b 10 (plusmn15)b 30 (plusmn19)b

reduction L3 0 889 915 745lowastDifferent small letters in rows indicate the existence of a statistical difference (119901 lt 001)

3 Results and Discussion

The results are shown in Table 1 which shows the meanstandard deviation and the H contortus larvae reductionpercentage after being recovered from pots after 12 days ofinteraction between nematophagous fungi and nematode lar-vae The average temperature value during the experimentalperiodwas 161∘C ranging from 15 to 33∘C the environmentalrelative humidity was 7103 ranging from 152 to 108 and7mmof rainThese environmental conditions are consideredas ideal for the development of ruminant parasitic larvae [22]and nematophagous fungi [23]

The results obtained using D flagrans in reducing thenematode larvae population into animal faeces have gen-erated the development of studies focused on the produc-tion of chlamydospores which are resistant stages of somemicrofungi for the control of ruminant parasitic nematodes[13] The results of the present work are promising andan important reduction in the larval population recoveredfrom treated pots has shown evidence that fungi producesatisfactory results with the use of 5 times 106 conidia andchlamydospores per pot and although this is only a modelof study it gives a clear image about the potential use ofthis biological system of control of infective larvae on thecontaminated grass

The recovery larvae mean values have shown statisticaldifference among the treated groups when compared withcontrol group (119901 lt 0001) However no differences werefound among the treated groups (119901 gt 0005) (Table 1) Suchresults corroborate those published of De Almeida et al [24]that demonstrated high effectiveness of the specieD flagransagainst different genera of parasites affecting ruminants suchas Haemonchus Teladorsagia and Trichostrongylus

The nematophagous C rosea fungus was able to reduceby 889 the number H contortus L

3 D flagrans was able to

reduce by 915 the number H contortus L3 The association

of the micro fungiD flagrans and C rosea was able to reduce745 of H contortus (L

3) infective larvae

Higher in vitro reductions (9421ndash9961) in the Hcontortus L

3population by different D flagrans isolates were

recently found by Priego-Cortes [25] although it is importantto remark that this research was performed under in vitroconditions on water agar plates In the same work the invitro activity ofD flagrans isolates FTHO-8M3 andDFIPCshowed 992 9823 and 988 reductions against the root-knot nematodeMeloidogyne sp Such results are encouragingin searching for a fungal candidate as a potential agent ofagricultural pest control

Assis et al [5] evaluated the predatory activity of Dflagrans and M thaumasium in the control of nematodiasis

in grazing cattle by administering fungal pellets obtaininga reduction of 478 and 5667 of the number of larvaerespectively and these results were lower than those foundin our study although conducted in animals grazing

In the present study the nematophagous fungus C roseareduced by 769 the number of H contortus L

3under envi-

ronmental conditions Similar results were recently obtainedusing two isolates C rosea (Yucatan strain) and Clonostachyssp

One Campeche strain C rosea reduced 842 and 595Hcontortus L

3 respectively These fungi also showed 943 and

959 reduction on the phytonematodeMeloidogyne sp (J2)respectively [26]

Baloyi et al [27] evaluated the in vitro predatory activityof C rosea (in faeces and in water) against trichostrongylidslarvae and they reported 699 and 893 reductions respec-tively of the number of trichostrongylids larvae in bioassaysmade in sheep feces andwaterOn the other hand Rodriguez-Martınez [28] evaluated in vitro predatory activity of C roseaagainst Rhabditis sp Caenorhabditis elegans Panagrellusredivivus Butlerius sp and H contortus and they recorded719 947 927 100 and 877 larvae reductions respectivelyTheir results have shown evidence about the potential of thesespecies in the control of parasites of veterinary importanceThese results are similar to those described in the presentwork performed in soil pots

Dong et al [10] showed that the metabolites obtainedof C rosea were lethal 24 hours after treatment to 50of C elegans P redivivus and B xylophilus larvae in ourwork we obtained better results of predation for free-livingnematodes same test being done in vivo Other researchesin phytopathology highlight the relevance and efficiency ofthe speciesC rosea as entomopathogenic againstCicadellidaehemiptera causing respectively 825 and 45 mortality inOncometopia tucumana and Tapajosa rubromaginata at 14days after incubation [29] Vega et al [30] showed that Crosea and B bassiana associated were able to reduce 825of the insects Hypothenemus hampei in coffee beans andCarreno-Perez et al [31] showed that C rosea reduction 79of diseases caused by plant pathogenic fungus Phytophthoracactorum isolated on apple fruits

According to Ayers et al [32] a group of compoundsextracted from C candelabrum showed 90 of efficacyagainst H contortus These compounds were synthesized byRama Rao [11] the macrolide clonostachydiol derived fromC cilindrospora that showed 80ndash90 of reduction of Hcontortus in artificially infected sheep when subcutaneouslyadministered at a dose of 25mgkg corroborating with 21our results although we have worked with environmentalcontrol


The association of the micro fungi D flagrans and Crosea was able to reduce 745 of H contortus (L

3) infective

larvae Furthermore Baloyi et al [27] demonstrated thatthe combination of B thuringiensis and C rosea showedan additive effect in vitro causing a mortality of 767 ofparasites nematode of sheep possibly due to the action of Crosea (enzyme production) and B thuringiensis (endotoxinproduction)

Some studies have noted the combination ofnematophagous fungi under natural conditions to combatparasitic forms (eggs andor larvae) of gastrointestinalnematode parasites This association of fungi has been testedand the results are relevant Tavela et al (2013) reported thatthe nematophagous fungi have been used as an alternativefor controlling gastrointestinal nematodes from domesticanimals under natural and laboratory conditions At thattime those authors demonstrated that the associations ofD flagrans and M thaumasium were viable after passingthrough the gastrointestinal tract in horses and could beused under natural conditions However these authors havestated that it is unclear ldquowhether the association of some ofthese species could bring some kind of advantagerdquo from thebiological point of view since the antagonism or synergismbetween species must be better understood

In the present study the association of Clonostachys andD flagranswas effective and can be used in new experimentaldesigns under laboratory conditions and in the field Crosea and D flagrans have shown nematicidal effectivenesswhen were used alone or in combination effectively reducingthe population of infective larvae (L

3) of H contortus in

controlled environmental conditionsHowever the authors believe that there is a need for

further studies to determine the existence of additive syner-gistic or antagonistic effects between these species It is readhere that Braga et al (2014) discuss the predatory activityof an isolate from the nematophagous fungus Arthrobotrysrobusta as an example of predatory activity on L

3of H con-

tortus At that time the authors demonstrated a percentagereduction of 738 by the end of seven days Thus in thisstudy it is suggested that the tested association (Clonostachys+ Duddingtonia) could be further explored in future studiesof biological control since the percentage obtained was745 This is the first report of C rosea and D flagransassociation and is the first report to control H contortus inenvironmental conditions

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

Theauthors alsowould like to thankCNPq Capes Fapes andFapemig for financial support and grant concession

References

[1] F Pisseri C Benedictis S P Roberti and B Azzarello ldquoSus-tainable animal production systemic prevention strategies in

parasitic diseases of ruminantsrdquo Alternative and IntegrativeMedicine vol 2 no 2 2013

[2] A Al-Rofaai W A Rahman and M Abdulghani ldquoSensitivityof two in vitro assays for evaluating plant activity against theinfective stage of Haemonchus contortus strainsrdquo ParasitologyResearch vol 112 no 2 pp 893ndash898 2013

[3] F R Braga and J V de Araujo ldquoNematophagous fungi forbiological control of gastrointestinal nematodes in domesticanimalsrdquo Applied Microbiology and Biotechnology vol 98 no1 pp 71ndash82 2014

[4] A S Cezar J B Catto and I Bianchin ldquoControle alternativode nematodeos gastrintestinais dos ruminantes atualidade eperspectivasrdquo Ciencia Rural vol 38 no 7 pp 2083ndash2091 2008

[5] R C L Assis F D Luns J V Araujo et al ldquoComparisonbetween the action of nematode predatory fungi Duddingtoniaflagrans and Monacrosporium thaumasium in the biologicalcontrol of bovine gastrointestinal nematodiasis in tropicalsoutheastern Brazilrdquo Veterinary Parasitology vol 193 no 1ndash3pp 134ndash140 2013

[6] P Mendoza-de-Gives and J F J Torres-Acosta ldquoBiotechnologi-cal use of fungi in the control of ruminant parasitic nematodesrdquoin Fungi Types Environmental Impact and Role in Disease VArias and A Paz-Silva Eds Nova Science Publishers NewYork NY USA 2012

[7] F R Braga J V Araujo F E F Soares et al ldquoInteraction of thenematophagous fungus Duddingtonia flagrans on Amblyommacajannense engorged females and enzymatic characterisation ofits chitinaserdquo Biocontrol Science and Technology vol 23 no 5pp 584ndash594 2013

[8] V L R Vilela T F Feitosa F R Braga et al ldquoBiological controlof goat gastrointestinal helminthiasis by Duddingtonia flagransin a semi-arid region of the northeastern Brazilrdquo VeterinaryParasitology vol 188 no 1-2 pp 127ndash133 2012

[9] J Li J Yang X Huang and K-Q Zhang ldquoPurification andcharacterization of an extracellular serine protease fromClonos-tachys rosea and its potential as a pathogenic factorrdquo ProcessBiochemistry vol 41 no 4 pp 925ndash929 2006

[10] J-Y Dong H-P He Y-M Shen and K-Q Zhang ldquoNematici-dal epipolysulfanyldioxopiperazines fromGliocladium roseumrdquoJournal of Natural Products vol 68 no 10 pp 1510ndash1513 2005

[11] A V Rama Rao V S Murthy and G V M Sharma ldquoStudiesdirected towards the synthesis of clonostachydiol Part IrdquoTetrahedron Letters vol 36 no 1 pp 139ndash142 1995

[12] M Faedo M Larsen and S Thamsborg ldquoEffect of differenttimes of administration of the nematophagous fungusDudding-tonia flagrans on the transmission of ovine parasitic nematodeson pasturemdasha plot studyrdquo Veterinary Parasitology vol 94 no1-2 pp 55ndash65 2000

[13] A Paz-Silva I Francisco R O Valero-Coss et al ldquoAbility of thefungusDuddingtonia flagrans to adapt to the cyathostomin egg-output by spreading chlamydosporesrdquo Veterinary Parasitologyvol 179 no 1ndash3 pp 277ndash282 2011

[14] N F Ojeda-Robertos J F J Torres-Acosta A J Ayala-BurgosC A Sandoval-Castro R O Valero-Coss and P Mendoza-de-Gives ldquoDigestibility of Duddingtonia flagrans chlamydosporesin ruminants In vitro and in vivo studiesrdquo BMC VeterinaryResearch vol 5 article 46 2009

[15] J K Chamuah P Perumal and A Mech ldquoEpidemiology andcontrol measures of helminth parasites in small ruminantsrdquoInternational Journal of Bio-Resource and Stress Managementvol 4 pp 278ndash287 2013


[16] E Jagla E Jodkowska and M Popiolek ldquoAlternative methodsfor the control of gastrointestinal parasites in horses with aspecial focus on nematode predatory fungi a reviewrdquo Annalsof Animal Science vol 13 no 2 pp 217ndash227 2013

[17] M Larsen ldquoBiological control of nematode parasites in sheeprdquoJournal of Animal Science vol 84 pp E133ndashE139 2006

[18] G L BarronTheNematode-Destroying Fungi Topics inMycol-ogy no 1 Canadian Biological Publications Ontario Canada1977

[19] R D Llerandi-Juarez and P Mendoza-de Gives ldquoResistanceof chlamydospores of nematophagous fungi to the digestiveprocesses of sheep inMexicordquo Journal of Helminthology vol 72no 2 pp 155ndash158 1998

[20] E Liebano-Hernandez ldquoIdentificacion morfometrica de larvasinfectantes de nematodos gastrointestinales y pulmonares enrumiantes domesticos de Mexicordquo in Diagnostico y control delos nematodos gastrointestinales de los rumiantes en Mexico VM V Prats Ed pp 27ndash34 2nd edition 2004

[21] A Chaney Identification of Internal Parasites of Sheep andGoats Honors College 2012

[22] E Liebano-Hernandez ldquoEcologıa de larvas de nematodos gas-trointestinales de bovinos ovinos y caprinosrdquo in Epidemiologıade Enfermidades Parasitarias em Animales Domesticos E QRomero J A F Castillo F I Velarde and M E L ArellanoEds Version Electronic 1st edition 2011

[23] O D Dhingra and J B Sinclair Basic Plant Pathology MethodsCRC Press 1995

[24] G L De Almeida J M Santurio J O J Filho et al ldquoPredatoryactivity of the fungusDuddingtonia flagrans in equine strongyleinfective larvae on natural pasture in the Southern Region ofBrazilrdquo Parasitology Research vol 110 no 2 pp 657ndash662 2012

[25] I Priego-Cortes Aislamiento y caracterizacion morfologica ymolecular del hongo nematofago Duddingtonia flagrans y eval-uacion de su actividad biologica in vitro en contra de nematodosde importancia para la industria agrıcola y pecuaria [Tesis deLicenciatura] Universidad Politecnica del Estado de Morelos2013

[26] S Rojas-Jimenez Evaluacion de la actividad nematicida in vitrodel hongo Clonostachys spp en contra de Haemonchus contortus(L3) y Meloidogyne sp (J2) [Tesis de Licenciatura] UniversidadPolitecnica del Estado de Morelos 2013

[27] M A Baloyi M D Laing and K S Yobo ldquoIsolation and invitro screening of Bacillus thuringiensis and Clonostachys roseaas biological control agents against sheep nematodesrdquo AfricanJournal of Agricultural Research vol 6 no 22 pp 5047ndash50542011

[28] R Rodriguez-Martınez Evaluacion de la actividad letal in vitrodel hongo nematofagoClonostachys rosea contra cinco nematodosdiferentes [Tesis de Licenciatura] Facultad de Medicina Veteri-naria y Zootecnia Universidad del Estado de Mexico 2011

[29] A V Toledo E Virla R A Humber S L Paradell and CC L Lastra ldquoFirst record of Clonostachys rosea (AscomycotaHypocreales) as an entomopathogenic fungus of Oncometopiatucumana and Sonesimia grossa (Hemiptera Cicadellidae) inArgentinardquo Journal of Invertebrate Pathology vol 92 no 1 pp7ndash10 2006

[30] F E Vega F Posada M Catherine Aime M Pava-Ripoll FInfante and S A Rehner ldquoEntomopathogenic fungal endo-phytesrdquo Biological Control vol 46 no 1 pp 72ndash82 2008

[31] A J Carreno Perez J O Blanco Valbuena and B VillegasEstrada ldquoSeleccion de hongos biocontroladores de Phytoph-thora cactorum agente causal de la pudricion radical y decorona en manzanordquo Agronomia vol 14 pp 89ndash96 2006

[32] S Ayers D L Zink K Mohn et al ldquoAnthelmintic constituentsof Clonostachys candelabrumrdquo Journal of Antibiotics vol 63 no3 pp 119ndash122 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


methods of control is biological control which has showna good effectiveness either in laboratory trials or in pastureexperiments which is an important activity reducing thefree-living stages of mayor gastrointestinal helminths Thismethod can be considered as a useful tool of control whichreduces the economic losses caused by parasites and canpromote a higher animal productivity [7 8]

The species Clonostachys rosea belonging to the orderHypocreales (family Bionectriacea) has being recorded as aparasite of other microfungi and also as a nematode parasiteapart from being a saprophyte fungus [9] This speciescolonizes plants and it has attracted interest by workers as apotential biological control agent since it produces volatileorganic compounds toxic for some living organisms [10 11]

Duddingtonia flagrans is an ldquoold friendrdquo of scientists whoenvision biological control as an alternative to the treatmentof herd animals by chemical means and also in the futureof prevention of high levels of contamination by nematodesin public areas and squares used by humans [3 12] Again itmust be remembered here that the ability to pass through thegastrointestinal tract of domestic animals makes this speciesof fungus an eternal ally in combating nematodes present inthe environment [13]

This fungal species is said to be the ldquomost promisingrdquo Itsmain focus of activity culminates in the predation of infectivehelminth forms (in this case nematode larvae) by means ofsimple adhesive hyphae

The fungus D flagrans belonging to the order Monil-iales (family Moniliaceae) is one of the most promisingmicroorganisms for biological control of ruminant parasiticnematodes [14] This species is harmless to plants animalsand human being [15 16] and it is possible to isolate it mainlyfrom soil samples and animal faeces [17]Thismicroorganismpossesses dual food habits being saprobius in absence ofnematodes and becoming either predatory or parasite ofnematodes in the presence of them [18]

Taking into account the representation of the worldlivestock and its socioeconomics it is important to evaluateimprove and provide the use of technologies that can con-tribute to the development of this segment of agrobusinessThe aim of this study was to evaluate the predatory activity offungal isolates of the speciesC rosea andD flagrans and theirassociation onH contortus infective larvae (L

3) inmicroplots

with graminaceous forage plant

2 Material and Methods

21 Fungi TheC roseaYucatan strain belonging to theCICYcollection was usedThis strain was maintained in 2 water-agar (2WA) at the Department of Helminthology (CENID-PAVET-INIFAP-MEXICO) The D flagrans fungal strain(FTHO-8) belonging to the Fungal Collection of CENID-Parasitologıa Veterinaria INIFAP-Mexico was used Thisstrain was originally obtained from a sheep faecal samplefrom a farm at Fierro del ToroVillage HuitzilacMunicipalityMorelos State Mexico [19]

22 Nematodes A H contortus isolate was originally ob-tained from a naturally infected sheep from ldquoLas Margaritasrdquo

Experimental Sheep Farm (INIFAP) inHueytamalcoMunici-pality in the state of PueblaMexico AH contortus egg donorlambwas orally infectedwith an aqueous suspension contain-ing 350 larvaekg of body weight Twenty days after infectionthe presence of H contortus eggs in faecal samples wasdetected through the McMaster technique Faecal cultureswere prepared by mixing faeces with polystyrene particles inplastic bowls Water was added to the faecal cultures and themixture was homogenized to keep the cultures hydrated andoxygenated to promote better larval hatching [20] Faecal cul-tures were covered with paper foil and incubated for 7 days atroom temperature (25ndash30∘C) Infective larvae were extractedfrom faeces using the Baermann funnel technique [21] Thelarvae were washed several times in density gradients of 50sucrose solution and rinsed and suspended in sterile water

23 Assay The pots were conformed as follows thirty-sixplastic (gardening) pots with a capacity of 100 grams (75times 55mm) were used Seventy grams of soil acquired fromcommercial nurseries was autoclaved at 12 atm and 115∘Cfor 15 minutes and added to the plastic pots Every pot withsoil was added with 006 grams of a commercial mixture ofdifferent generaspecies grass seeds about 70 graminaceousseeds were added to the soil surface in order to obtain ahomogeneous grass growing Six mL of top water was addedto every pot every day during 18 days of the experiment usinga Pasteur pipette to maintain the pots humidity

After germination the pots were randomly divided intofour (4) experimental groups with 8 replicates each Allgroups were added with 10mL average of an acqueos sus-pension with 618 H contortus L

3approximately Group 1 was

used as control and only received the infective larvae Groups2 and 3 received D flagrans chlamydospores and C roseaconidia at doses of 5 times 106 respectively Group 4 received thecombination of 5 times 106 D flagrans chlamydospores + 5 times 106C rosea conidia

The pots were maintained under a shading rate hedgingprotected screen and after 12 days of interaction betweennematophagous fungi and parasitic nematodes the wholenematode population from total soil and grass of every singlepot was recovered using the Baermann Funnel method [21]

24 Location The experiment was conducted in CENID-PAVET-INIFAP-MEXICO located at 18∘5310158400410158401015840N latitudeand 98∘5110158403410158401015840W and 161516 meters above sea level in themunicipality of Jiutepec Morelos Mexico

25 Weather Data Meteorological data including maximumand minimum temperatures relative humidity and rain-fall during the experiment were obtained from the closestexperimental weather station (National Institute for WaterTechnology) Jiutepec Morelos Mexico

26 Statistical Analysis Datawere analysed using anANOVAtest and the Tukey test was used as a complementary tech-nique for discriminating the mean different from the othersData were analysed using the software BioEstat 53













3) infective larvae







3under envi-










3) infective







3of H con-




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













3) infective larvae







3under envi-










3) infective







3of H con-




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3) infective







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Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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