Biology, growth parameters and enzymatic activity of Pochonia chlamydosporia isolated from potato cyst and root-knot nematodes

in Nematology
Restricted Access
Get Access to Full Text
Rent on DeepDyve

Have an Access Token?



Enter your access token to activate and access content online.

Please login and go to your personal user account to enter your access token.



Help

Have Institutional Access?



Access content through your institution. Any other coaching guidance?



Connect

Pochonia chlamydosporia, a widespread fungal parasite of potato cyst nematodes (PCN), Globodera spp., and root-knot nematodes (RKN), Meloidogyne spp., has been studied as a biological control agent. Three Portuguese isolates (Pc1, Pc2, Pc3) obtained from PCN eggs and two non-native isolates (Vc10, Pc280) were characterised using ERIC-PCR and screened by in vitro assays for their ability to produce chlamydospores, parasitise eggs of Globodera rostochiensis and Meloidogyne chitwoodi and colonise the rhizosphere of barley. The effects of temperature on growth, sporulation, parasitism and enzymatic activity were also evaluated. Isolates Pc1 and Pc3, despite their different geographical origins, had identical molecular profiles. Pc2 produced the higher numbers of chlamydospores in solid medium (1.15 × 107 chlamydospores g−1), whereas Pc3 produced the least (3 × 105 chlamydospores g−1). These isolates extensively colonised the rhizosphere of barley (>90% root fragments) and the proportion of parasitised eggs, detected on agar plates, was low (<60% for RKN and <55% for PCN), Pc1 being the best parasite against both nematode species. The influence of temperature was similar for all isolates: no growth was observed at 10, 33 and 35°C. Spores/hyphal fragments remained viable for nearly 1 month at 10 and 33°C and isolates resumed growth after incubation at 25°C, although chlamydospores and conidia production, viability and nematode egg parasitism were affected. Exposure to 35°C was lethal for isolates Pc1, Pc2 and Pc280. When grown in liquid media all isolates produced esterases, but protease activity was only observed in Pc2 and Vc10. The highest enzymatic activity was detected in isolate Pc2 in low nutrient media. Enzymatic activity decreased in the presence of nematode eggs for both Pc2 and Vc10. Molecular, biochemical and biological analyses, including biotic and abiotic factors, are important in the screening of potential biological control agents, particularly in the case of P. chlamydosporia, due to the high variability among isolates.

Biology, growth parameters and enzymatic activity of Pochonia chlamydosporia isolated from potato cyst and root-knot nematodes

in Nematology

Sections

References

AbrantesI.SantosS.BourneJ.CiancioA.Lopez-LlorcaL.KerryB.MotaM.TzortzakakisE.Verdejo-LucasS. (2002). A manual for research on Verticillium chlamydosporium a potential biological control agent for root-knot nematodes. KerryB.R.BourneJ. (Eds). Ghent, BelgiumInternational Organization for Biological and Integrated Control of Noxious Animals and Plants/West Palearctic Regional Section (IOBC/WPRS)84 pp.

AroraD.K.HirschP.R.KerryB.R. (1996). PCR-based molecular discrimination of Verticillium chlamydosporium isolates. Mycological Research 100801-809.

BourneJ.M.KerryB.R. (2000). Observations on the survival and competitive ability of the nematophagous fungus Verticillium chlamydosporium in soil. International Journal of Nematology 109-18.

BourneJ.M.KerryB.R.de LeijF.A.A.M. (1996). The importance of the host plant on the interaction between root-knot nematodes (Meloidogyne spp.) and the nematophagous fungus, Verticillium chlamydosporium Goddard. Biocontrol Science and Technology 6539-548.

CarderJ.H.SegersR.ButtM.T.BarbaraD.J.von MendeN.CoosemansJ. (1993). Taxonomy of the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium based on secreted enzyme activities and RFLP analysis. Journal of Invertebrate Pathology 62178-184.

da ConceiçãoI.L.P.M.da CunhaM.J.M.FeioG.CorreiaM.dos SantosM.C.V.de O. AbrantesI.M.SantosM.S.N.A. (2009). Root-knot nematodes, Meloidogyne spp., on potato in Portugal. Nematology 11311-313.

da CunhaM.J.M.da ConceiçãoI.L.P.M.de O. AbrantesI.M.EvansK.de A. SantosM.S.N. (2004). Characterisation of potato cyst nematodes from Portugal. Nematology 655-58.

De LeijF.A.A.M.KerryB.R. (1991). The nematophagous fungus Verticillium chlamydosporium as a potential biological control agent for Meloidogyne arenaria. Revue de Nématologie 14157-164.

EstevesI.PeteiraB.AtkinsS.MaganN.KerryB. (2009). Production of extracellular enzymes in different isolates of Pochonia chlamydosporia. Mycological Research 113867-876.

Flores-CamachoR.AtkinsS.D.Manzanilla-LópezR.H.Cid del Prado-VeraI.Martínez-GarzaA. (2008). Aislamientos Mexicanos de Pochonia chlamydosporia var. chlamydosporia (Goddard) Gams y Zare para el Control Biológico de Nacobbus aberrans (Thorne, 1935) Thorne y Allen, 1944. Revista Mexicana de Fitopatologia 2693-104.

Hidalgo-DiazL.BourneJ.M.KerryB.R.RodriguezM.G. (2000). Nematophagous Verticillium spp. in soils infested with Meloidogyne spp. in Cuba: isolation and screening. International Journal of Pest Management 46277-284.

HirschP.MauchlineT.MendumT.KerryB. (2000). Detection of the nematophagous fungus Verticillium chlamydosporium in nematode-infested plant roots using PCR. Mycological Research 104435-439.

IrvingF.KerryB.R. (1986). Variation between strains of the nematophagous fungus Verticillium chlamydosporium Goddard II. Factors affecting parasitism of cyst nematode eggs. Nematologica 32474-485.

KerryB.R. (2000). Rhizosphere interactions and the exploitation of microbial agents for the biological control of plant-parasitic nematodes. Annual Review of Phytopathology 38423-441.

KerryB.R.HirschP.R. (2011). Ecology of Pochonia chlamydosporia in the rhizosphere at the population, whole organism and molecular scales. In: DaviesK.G.SpiegelY. (Eds). Biological control of plant-parasitic nematodes: building coherence between microbial ecology and molecular mechanisms. London, UKSpringer pp.  171-182.

KerryB.R.IrvingF.HornseyJ.C. (1986). Variation between strains of the nematophagous fungus Verticillium chlamydosporium Goddard: I. Factors affecting growth in vitro. Nematologica 32461-473.

López-LlorcaL.V.RobertsonW.M. (1992). Ultrastructure of infection of cyst nematode eggs by the nematophagous fungus Verticillium chlamydosporium. Nematologica 3965-74.

MaganN. (2001). Physiological approaches to improving the ecological fitness of fungal biocontrol agents in fungi as biocontrol agents. In: ButtT.JacksonC.MaganN. (Eds). Fungi as biocontrol agents – progress problems and potential. London, UKCABI Publishing pp.  239-251.

Manzanilla-LópezR.H.AtkinsS.D.ClarkI.M.KerryB.R.HirschP.R. (2009). Measuring abundance, diversity and parasitic ability in two populations of the nematophagous fungus Pochonia chlamydosporia var. chlamydosporia. Biocontrol Science and Technology 19391-406.

MarínS.SanchisV.SáenzR.RamosA.J.VinasI.MaganN. (1998). Ecological determinants for germination and growth of some Aspergillus and Penicillium spp. from maize grain. Journal of Applied Microbiology 8425-36.

Mendoza de GivesP.BehnkeJ.M.DaviesK.G. (2003). Extracellular enzyme production by nematophagous fungi in the presence and absence of nematodes. International Journal of Nematology 1327-36.

Morgan-JonesG.WhiteJ.F.Rodríguez-KábanaR. (1983). Phytonematode pathology: ultrastructural studies. I. Parasitism of Meloidogyne arenaria eggs by Verticillium chlamydosporium. Nematropica 13245-260.

MortonC.O.MauchlineT.H.KerryB.R.HirschP.R. (2003). PCR-based fingerprinting indicates host-related genetic variation in the nematophagous fungus Pochonia chlamydosporia. Mycological Research 107198-205.

NageshM.HussainiS.S.RamanujamB.RangeswaranR. (2007). Molecular identification, characterization, variability and infectivity of Indian isolates of the nematophagous fungus Pochonia chlamydosporia. Nematologia Mediterranea 3547-56.

NicolJ.M.TurnerS.J.CoyneD.L.den NijsL.HocklandS.Tahna MaafiZ. (2011). Current nematode threats to world agriculture. In: JonesJ.GheysenG.FenollC. (Eds). Genomics and molecular genetics of plant-nematode interactions. London, UKSpringer pp.  21-43.

Olivares-BernabeuC.M.López-LlorcaL.V. (2002). Fungal egg-parasites of plant-parasitic nematodes from Spanish soils. Revista Iberoamericana de Micologia 19104-110.

RossoL.C.Finetti-SialerM.M.HirschP.R.CiancioA.KerryB.R.ClarkI.A. (2011). Transcriptome analysis shows differential gene expression in the saprotrophic to parasitic transition of Pochonia chlamydosporia. Applied Microbiology and Technology 901981-1994.

SegersR.ButtT.M.KerryB.R.PeberdyJ.F. (1994). The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ. Microbiology 1402715-2723.

ShepherdA. (1986). Extraction and estimation of cyst nematodes. In: SoutheyJ.F. (Ed.). Laboratory methods for work with plant and soil nematodes. London, UKMinistry of Agriculture, Fisheries and Food, Her Majesty’s Stationery Office pp.  31-49.

St. LegerR.J.CooperR.M.CharnleyA.K. (1986). Cuticle-degrading enzymes of entomopathogenic fungi: regulation of production of chitinolytic enzymes. Journal of General and Applied Microbiology 1331509-1517.

StirlingG.R. (1991). Biological control of plant-parasitic nematodes: progress problems and prospects. Wallingford, UKCABI Publishing.

TikhonovV.E.López-LlorcaL.V.SalinasJ.JanssonH.-B. (2002). Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium. Fungal Genetics and Biology 3567-78.

TrudgillD.L.BlokV.C. (2001). Apomitic, polyphagous root-knot nematodes: exceptionally successful and damaging biotrophic root pathogens. Annual Review of Phytopathology 3953-77.

Figures

  • View in gallery

    Molecular characterisation of five Pochonia chlamydosporia isolates (Vc10, Pc280, Pc1, Pc2, Pc3). PCR product of DNA amplified with the β-tubulin primer set (A) and ERIC-PCR (B) profiles. nc: negative control without template DNA; M: DNA size marker Smart Ladder SF, 100 bp ladder (Eurogentec, Belgium). Empty lanes have been excised from the pictures to bring the marker and profiles together.

  • View in gallery

    A: Number of chlamydospores; B: Percentage of germinated chlamydospores; C: Percentage of colonised roots of five Pochonia chlamydosporia isolates (Pc1, Pc2, Pc3, Vc10, Pc280) grown for 25 days on 1.7% corn meal agar (CMA). Bars represent standard error of means. Columns with the same letter are not significantly different according to LSD test (P<0.05).

  • View in gallery

    A: Percentage of parasitised eggs; B: Number of conidia of five Pochonia chlamydosporia isolates (Pc1, Pc2, Pc3, Vc10, Pc280) grown for 25 days on 1.7% corn meal agar (CMA). Bars represent standard error of means. Columns with the same letter are not significantly different according to LSD test (P<0.05). Significantly different from same pattern columns according to LSD test (P<0.01).

  • View in gallery

    Dendograms showing similarities between groups based on extracellular enzymatic activity of five Pochonia chlamydosporia isolates (Pc1, Pc2, Pc3, Vc10 and Pc280) grown for: A: 10 days in Czapek Dox broth (Czpk) and 7 days in Czpk followed by 3 days in weak Czapek Dox broth (wCzpk); B: 7 days in Czpk followed by a transfer to wCzpk inoculated with 100 eggs ml−1 either of Globodera rostochiensis (wCzpk + PCN) or Meloidogyne chitwoodi (wCzpk + RKN). Results are the average values of enzymatic activity between two replicates of each isolate for the enzymes: α-galactosidase, acid phosphatise, esterase (C4), esterase lipase (C8), leucine arylamidase, N-acetyl-β-glucosaminidase, naphthol-AS-BI-phosphohydrolase, and valine arylamidase. The dendrograms were constructed using cluster analysis (group average).

Index Card

Content Metrics

Content Metrics

All Time Past Year Past 30 Days
Abstract Views 5 5 4
Full Text Views 4 4 4
PDF Downloads 1 1 1
EPUB Downloads 0 0 0