Biological control of plant-parasitic nematodes by the fungus Clonostachys rosea

Mudassir Iqbal

Biological control of plant-parasitic nematodes by the fungus Clonostachys rosea

Bidragets översatta titel: Biologisk bekämpning av växtparaitiska nematoder med svampen Clonostachys rosea

Mudassir Iqbal

Institutionen för skoglig mykologi och växtpatologi

Publikation: Avhandling › Doktorsavhandling

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Sammanfattning

Sjukdomar på växter som orsakas av växtparasitiska nematoder är ett allvarligt hot mot en hållbar växtproduktion genom höga skördeförluster och brist på effektiva växtskyddsmetoder. Biologisk bekämpning är en lovande metod för att minska skördeförluster orsakade av växtparasitiska nematoder. I den här studien undersöktes hur svampen Clonostachys rosea isolat IK726 påverkar nematod-populationer i en naturligt infekterad jord i ett försök med vete i klimatkammare med kontrollerad miljö. Populationerna av växtparasitiska nematoder i jorden minskade med 40 till 73% när C. rosea applicerades jämfört med jord utan C. rosea. Icke-växtparasitiska nematoder påverkades inte. Applicering av C. rosea i jorden ökade vikten och längden på veteplantorna med 20 respektive 24%. Undersökningar av interaktionen mellan C. rosea och nematoder med mikroskop resulterade inte i några bevis för direkt parasitism. Filtrat av potatis dextros vätskekulturer från C. rosea uppvisade dock toxicitet mot nematoder. En jämförande analys av proteas-gener i svampars arvsmassa visade att C. rosea innehåller fler proteas-gener än andra svampar som används för biologisk bekämpning. En evolutionär analys av genfamiljer av proteaser visade att underfamiljerna S8A, S9X och S33 innehåller höga antal gener i C. rosea, vilket indikerar att dessa proteaser har en viktig funktion i biotiska interaktioner. En storskalig associationsanalys av 53 isolat av C. rosea identifierade vidare 279 mutationer som segregerade med in vitro antagonism mot växtparasitiska nematoder. Två gener (nps4 och nps5) för icke-ribosomala peptidsyntaser (NRPS) identifierades i de genomiska regioner som segregerade med antagonism, och ytterligare en NRPS gen (nps1) valdes ut för fortsatta funktionella studier baserat på befintlig litteratur. Mutanter av C. rosea där nps1, nps4 respektive nps5 inaktiverats med genetisk transformering uppvisade högre tillväxt och högre konidiering, jämfört med vildtypen. Filtrat från vätskekulturer av C. rosea mutanterna hade minskad toxicitet mot nematoder jämfört med vildtypen efter 24 timmars inkubation. Kulturfiltraten uppvisade dock fortfarande en viss toxicitet mot nematoder jämfört med kontrollbehandlingen, vilket indikerade att andra toxiska ämnen eller enzymer (t.ex. proteaser) fortfarande producerades av mutanterna. Inaktiveringen av nps1, nps4 och nps5 resulterade också i minskad förmåga till biologisk bekämpning av rotsjukdom hos vete orsakad av växtparasitiska nematoder hos mutanterna, jämfört med vildtypen.Den här studien visar att C. rosea kan bekämpa sjukdomar orsakade av växtparasitiska nematoder och förbättra tillväxten hos växter. Dessa egenskaper kan potentiellt användas inom integrerat växtskydd och därmed bidra till minskad användning av kemiska växtskyddsmedel.

Bidragets översatta titel	Biologisk bekämpning av växtparaitiska nematoder med svampen Clonostachys rosea
Originalspråk	Engelska
Kvalifikation	Doktor i filosofi
Förlag	Swedish University of Agricultural Sciences
Tryckta ISBN	978-91-7760-462-4
Elektroniska ISBN	978-91-7760-463-1
Status	Publicerad - 2019

Nyckelord

NRPS
antagonism
antibiosis
biocontrol
fungi
nematodes
proteases

SLU-serie

Acta Universitatis Agriculturae Sueciae

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title = "Biological control of plant-parasitic nematodes by the fungus Clonostachys rosea",

abstract = "Plant diseases caused by plant-parasitic nematodes are serious constraints to sustainable crop production due to high yield losses, the persistent nature of these nematodes and a lack of efficient control methods. Biological control is a promising approach to reduce plant diseases caused by nematodes.This study investigated the effect of the fungus Clonostachys rosea strain IK726 on nematode populations in a naturally nematode-infested soil planted with wheat in a climate chamber under controlled conditions. Populations of plant-parasitic nematodes extracted from soil and roots were 40 to 73\% lower in soils when C. rosea was applied than in untreated soils, whereas non-parasitic nematodes were unaffected. Soil inoculation with C. rosea increased the shoot weight and shoot length of wheat plants by 20 and 24\%, respectively. Light microscopy of in vitro C. rosea{\textendash}nematode interactions did not reveal evidence of direct parasitism; however, culture filtrates of C. rosea grown in potato dextrose broth (PDB) exhibited toxicity towards nematodes and immobilized nematodes. A genome-wide analysis of protease genes showed that C. rosea contains more protease genes than other studied biocontrol fungi. A computational analysis of gene family evolution revealed a high gene copy number of serine protease subfamilies S8A, S9X and S33 in C. rosea, suggesting the involvement of these proteases in biotic interactions. A genome-wide association analysis of 53 strains of C. rosea further identified 279 single-nucleotide polymorphism markers that were significantly associated with the in vitro antagonism trait against plant-parasitic nematodes. Two non-ribosomal peptide synthetase (NRPS) genes (nps4 and nps5) were identified in genomic regions associated with nematicidal activity whereas nps1 was included based on previous published reports of functional studies. Gene deletion strains of nps1, nps4 and nps5 were generated and showed increased growth and conidiation rates. Culture filtrates from C. rosea Δnps1, Δnps4 and Δnps5 strains exhibited reduced nematicidal activity and immobilized lower numbers of nematodes compared with the wild type after 24 h of incubation. However, NRPS deletion strains still possessed some nematicidal activity compared with the PDB control treatment, which may be due to the presence of additional nematicidal compounds or enzymes (e.g., serine proteases). Furthermore, Δnps1, Δnps4 and Δnps5 strains showed reduced biocontrol efficacy in a naturally nematode-infested soil in a pot experiment and failed to reduce populations of nematodes in soil or in roots of wheat as efficiently as the wild type strain.This study demonstrates that C. rosea can control plant-parasitic nematodes and improve the growth of plants at the same time. The antagonistic potential of C. rosea could be used to control plant-parasitic nematodes, which may contribute to reduced applications of chemicals as part of an integrated pest management programme.",

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year = "2019",

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isbn = "978-91-7760-462-4",

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TY - BOOK

T1 - Biological control of plant-parasitic nematodes by the fungus Clonostachys rosea

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N2 - Plant diseases caused by plant-parasitic nematodes are serious constraints to sustainable crop production due to high yield losses, the persistent nature of these nematodes and a lack of efficient control methods. Biological control is a promising approach to reduce plant diseases caused by nematodes.This study investigated the effect of the fungus Clonostachys rosea strain IK726 on nematode populations in a naturally nematode-infested soil planted with wheat in a climate chamber under controlled conditions. Populations of plant-parasitic nematodes extracted from soil and roots were 40 to 73% lower in soils when C. rosea was applied than in untreated soils, whereas non-parasitic nematodes were unaffected. Soil inoculation with C. rosea increased the shoot weight and shoot length of wheat plants by 20 and 24%, respectively. Light microscopy of in vitro C. rosea–nematode interactions did not reveal evidence of direct parasitism; however, culture filtrates of C. rosea grown in potato dextrose broth (PDB) exhibited toxicity towards nematodes and immobilized nematodes. A genome-wide analysis of protease genes showed that C. rosea contains more protease genes than other studied biocontrol fungi. A computational analysis of gene family evolution revealed a high gene copy number of serine protease subfamilies S8A, S9X and S33 in C. rosea, suggesting the involvement of these proteases in biotic interactions. A genome-wide association analysis of 53 strains of C. rosea further identified 279 single-nucleotide polymorphism markers that were significantly associated with the in vitro antagonism trait against plant-parasitic nematodes. Two non-ribosomal peptide synthetase (NRPS) genes (nps4 and nps5) were identified in genomic regions associated with nematicidal activity whereas nps1 was included based on previous published reports of functional studies. Gene deletion strains of nps1, nps4 and nps5 were generated and showed increased growth and conidiation rates. Culture filtrates from C. rosea Δnps1, Δnps4 and Δnps5 strains exhibited reduced nematicidal activity and immobilized lower numbers of nematodes compared with the wild type after 24 h of incubation. However, NRPS deletion strains still possessed some nematicidal activity compared with the PDB control treatment, which may be due to the presence of additional nematicidal compounds or enzymes (e.g., serine proteases). Furthermore, Δnps1, Δnps4 and Δnps5 strains showed reduced biocontrol efficacy in a naturally nematode-infested soil in a pot experiment and failed to reduce populations of nematodes in soil or in roots of wheat as efficiently as the wild type strain.This study demonstrates that C. rosea can control plant-parasitic nematodes and improve the growth of plants at the same time. The antagonistic potential of C. rosea could be used to control plant-parasitic nematodes, which may contribute to reduced applications of chemicals as part of an integrated pest management programme.

AB - Plant diseases caused by plant-parasitic nematodes are serious constraints to sustainable crop production due to high yield losses, the persistent nature of these nematodes and a lack of efficient control methods. Biological control is a promising approach to reduce plant diseases caused by nematodes.This study investigated the effect of the fungus Clonostachys rosea strain IK726 on nematode populations in a naturally nematode-infested soil planted with wheat in a climate chamber under controlled conditions. Populations of plant-parasitic nematodes extracted from soil and roots were 40 to 73% lower in soils when C. rosea was applied than in untreated soils, whereas non-parasitic nematodes were unaffected. Soil inoculation with C. rosea increased the shoot weight and shoot length of wheat plants by 20 and 24%, respectively. Light microscopy of in vitro C. rosea–nematode interactions did not reveal evidence of direct parasitism; however, culture filtrates of C. rosea grown in potato dextrose broth (PDB) exhibited toxicity towards nematodes and immobilized nematodes. A genome-wide analysis of protease genes showed that C. rosea contains more protease genes than other studied biocontrol fungi. A computational analysis of gene family evolution revealed a high gene copy number of serine protease subfamilies S8A, S9X and S33 in C. rosea, suggesting the involvement of these proteases in biotic interactions. A genome-wide association analysis of 53 strains of C. rosea further identified 279 single-nucleotide polymorphism markers that were significantly associated with the in vitro antagonism trait against plant-parasitic nematodes. Two non-ribosomal peptide synthetase (NRPS) genes (nps4 and nps5) were identified in genomic regions associated with nematicidal activity whereas nps1 was included based on previous published reports of functional studies. Gene deletion strains of nps1, nps4 and nps5 were generated and showed increased growth and conidiation rates. Culture filtrates from C. rosea Δnps1, Δnps4 and Δnps5 strains exhibited reduced nematicidal activity and immobilized lower numbers of nematodes compared with the wild type after 24 h of incubation. However, NRPS deletion strains still possessed some nematicidal activity compared with the PDB control treatment, which may be due to the presence of additional nematicidal compounds or enzymes (e.g., serine proteases). Furthermore, Δnps1, Δnps4 and Δnps5 strains showed reduced biocontrol efficacy in a naturally nematode-infested soil in a pot experiment and failed to reduce populations of nematodes in soil or in roots of wheat as efficiently as the wild type strain.This study demonstrates that C. rosea can control plant-parasitic nematodes and improve the growth of plants at the same time. The antagonistic potential of C. rosea could be used to control plant-parasitic nematodes, which may contribute to reduced applications of chemicals as part of an integrated pest management programme.

KW - NRPS

KW - antagonism

KW - antibiosis

KW - biocontrol

KW - fungi

KW - nematodes

KW - proteases

KW - NRPS

KW - antagonism

KW - antibiosis

KW - biocontrol

KW - fungi

KW - nematodes

KW - proteases

UR - https://res.slu.se/id/publ/102565

M3 - Doctoral thesis

SN - 978-91-7760-462-4

T3 - Acta Universitatis Agriculturae Sueciae

PB - Swedish University of Agricultural Sciences

ER -