| 1. |
- Broberg, Martin, et al.
(author)
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Comparative genomics highlights the importance of drug efflux transporters during evolution of mycoparasitism in Clonostachys subgenus Bionectria (Fungi, Ascomycota, Hypocreales)
- 2021
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In: Evolutionary applications. - : Wiley. - 1752-4571. ; 14, s. 476-497
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Journal article (peer-reviewed)abstract
- Various strains of the mycoparasitic fungal speciesClonostachys roseaare used commercially as biological control agents for the control of fungal plant diseases in agricultural crop production. Further improvements of the use and efficacy ofC. roseain biocontrol require a mechanistic understanding of the factors that determines the outcome of the interaction betweenC. roseaand plant pathogenic fungi. Here, we determined the genome sequences of 11Clonostachysstrains, representing five species inClonostachyssubgenusBionectria, and performed a comparative genomic analysis with the aim to identify gene families evolving under selection for gene gains or losses. Several gene families predicted to encode proteins involved in biosynthesis of secondary metabolites, including polyketide synthases, nonribosomal peptide syntethases and cytochrome P450s, evolved under selection for gene gains (p <= .05) in theBionectriasubgenus lineage. This was accompanied with gene copy number increases (p <= .05) in ATP-binding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters predicted to contribute to drug efflux. MostClonostachysspecies were also characterized by high numbers of auxiliary activity (AA) family 9 lytic polysaccharide monooxygenases, AA3 glucose-methanol-choline oxidoreductases and additional carbohydrate-active enzyme gene families with putative activity (or binding) towards xylan and rhamnose/pectin substrates. Particular features of theC. roseagenome included expansions (p <= .05) of the ABC-B4 multidrug resistance transporters, the ABC-C5 multidrug resistance-related transporters and the 2.A.1.3 drug:H + antiporter-2 MFS drug resistance transporters. The ABC-G1 pleiotropic drug resistance transporter geneabcG6inC. roseawas induced (p <= .009) by exposure to the antifungalFusariummycotoxin zearalenone (1121-fold) and various fungicides. Deletion ofabcG6resulted in mutants with reduced (p < .001) growth rates on media containing the fungicides boscalid, fenhexamid and iprodione. Our results emphasize the role of biosynthesis of, and protection against, secondary metabolites inClonostachyssubgenusBionectria.
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| 2. |
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| 3. |
- Iqbal, Mudassir, et al.
(author)
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Bee-Vectored Aureobasidium pullulans for Biological Control of Gray Mold in Strawberry
- 2022
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In: Phytopathology. - 0031-949X .- 1943-7684. ; 112, s. 232-237
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Journal article (peer-reviewed)abstract
- Gray mold caused by Botrytis cinerea is a common postharvest disease in strawberries, reducing shelf life considerably. We investigated the potential of the yeast-like biocontrol fungus Aureobasidium pullulans (AP-SLU6) vectored by bumblebees (Bombus terrestris) in the Flying DoctorsVR system to inhibit the pathogen and increase the shelf life of harvested strawberries (cultivar Sonata). Using bumblebees as vectors of various biocontrol agents is becoming increasingly popular, but any potentially negative effects on bee performance have been understudied. Our results show that, over the 4-week period of the trial, the performance and activity of the bees were not negatively affected by A. pullulans. The bees successfully picked up the powder formulation; then, they carried and deposited it on the flowers. The vectoring of the biocontrol agent significantly reduced gray mold development on the harvested fruits by 45% and increased shelf life by 100% in comparison with control treatments. This suggests that the biocontrol fungus applied during flowering successfully reduced Botrytis infection and thus, effectively protected the fruits from gray mold. In addition, the bee-vectored application of the biocontrol agent was found to be significantly more effective than spray application because the latter may temporarily increase humidity around the flower, thereby creating a suitable environment for the pathogen to thrive. In summary, our study demonstrates that A. pullulans vectored by bumblebees can decrease gray mold infection and improve the shelf life of strawberries without adversely affecting the bees, thus providing a basis for the sustainable and efficient control of gray mold on strawberry.
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| 4. |
- Iqbal, Mudassir
(author)
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Biological control of plant-parasitic nematodes by the fungus Clonostachys rosea
- 2019
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Doctoral thesis (peer-reviewed)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–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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| 5. |
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| 6. |
- Iqbal, Mudassir, et al.
(author)
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Biological control of strawberry crown rot, root rot and grey mould by the beneficial fungus Aureobasidium pullulans
- 2021
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In: BioControl. - : Springer Science and Business Media LLC. - 1386-6141 .- 1573-8248. ; 66, s. 535-545
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Journal article (peer-reviewed)abstract
- Utilization of biocontrol agents is a sustainable approach to reduce plant diseases caused by fungal pathogens. In the present study, we tested the effect of the candidate biocontrol fungus Aureobasidium pullulans (De Bary) G. Armaud on strawberry under in vitro and in vivo conditions to control crown rot, root rot and grey mould caused by Phytophthora cactorum (Lebert and Cohn) and Botrytis cinerea Pers, respectively. A dual plate confrontation assay showed that mycelial growth of P. cactorum and B. cinerea was reduced by 33-48% when challenged by A. pullulans as compared with control treatments. Likewise, detached leaf and fruit assays showed that A. pullulans significantly reduced necrotic lesion size on leaves and disease severity on fruits caused by P. cactorum and B. cinerea. In addition, greenhouse experiments with whole plants revealed enhanced biocontrol efficacy against root rot and grey mould when treated with A. pullulans either in combination with the pathogen or pre-treated with A. pullulans followed by inoculation of the pathogens. Our results demonstrate that A. pullulans is an effective biocontrol agent to control strawberry diseases caused by fungal pathogens and can be an effective alternative to chemical-based fungicides.
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| 7. |
- Iqbal, Mudassir, et al.
(author)
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Biological control of strawberry diseases by Aureobasidium pullulans and sugar beet extract under field conditions
- 2023
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In: Journal of plant pathology. - 1125-4653 .- 2239-7264. ; 105, s. 933-941
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Journal article (peer-reviewed)abstract
- Grey mould (caused by Botrytis cinerea) is the most important pathogen underlying high fungicide dependence in strawberry fields. Reliable biocontrol agents (BCAs) with improved efficiency are needed to replace fungicides. The yeast-like beneficial fungus Aureobasidium pullulans (AP-SLU6) has previously exhibited great potential to combat grey mould in greenhouse environments. Here we report results from a two-year full-factorial field trial in a conventional strawberry field, in which we tested two different concentrations of A. pullulans (10(7) CFU/ml and 10(6) CFU/ml) and sugar beet extract (SBE). The results showed that all the field treatments reduced grey mould severity postharvest and increased shelf life of the harvested fruit in both years. The best effect was achieved using the highest conidial concentration of A. pullulans, which also resulted in 53% higher fruit production compared to the control treatment at the end of the season, indicating a plant-growth promoting effect of the BCA. These results reveal that spray applications of these novel BCAs contribute to reliable biocontrol of grey mould, leading to improvement of the shelf life of strawberry sales boxes. These findings suggest that A. pullulans and SBE can contribute to a shift from chemical fungicides to sustainable methods without compromising cropping security.
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| 8. |
- Iqbal, Mudassir, et al.
(author)
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Comparative evolutionary histories of fungal proteases reveal gene gains in the mycoparasitic and nematode-parasitic fungus Clonostachys rosea
- 2018
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In: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 18
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Journal article (peer-reviewed)abstract
- Background: The ascomycete fungus Clonostachys rosea (order Hypocreales) can control several important plant diseases caused by plant pathogenic fungi and nematodes. Subtilisin-like serine proteases are considered to play an important role in pathogenesis in entomopathogenic, mycoparasitic, and nematophagous fungi used for biological control. In this study, we analysed the evolutionary histories of protease gene families, and investigated sequence divergence and regulation of serine protease genes in C. rosea.Results: Proteases of selected hypocrealean fungal species were classified into families based on the MEROPS peptidase database. The highest number of protease genes (590) was found in Fusarium solani, followed by C. rosea with 576 genes. Analysis of gene family evolution identified non-random changes in gene copy numbers in the five serine protease gene families S1A, S8A, S9X, S12 and S33. Four families, S1A, S8A, S9X, and S33, displayed gene gains in C. rosea. A gene-tree / species-tree reconciliation analysis of the S8A family revealed that the gene copy number increase in C. rosea was primarily associated with the S08.054 (proteinase K) subgroup. In addition, regulatory and predicted structural differences, including twelve sites evolving under positive selection, among eighteen C. rosea S8A serine protease paralog genes were also observed. The C. rosea S8A serine protease gene prs6 was induced during interaction with the plant pathogenic species F. graminearum.Conclusions: Non-random increases in S8A, S9X and S33 serine protease gene numbers in the mycoparasitic species C. rosea, Trichoderma atroviride and T. virens suggests an involvement in fungal-fungal interactions. Regulatory and predicted structural differences between C. rosea S8A paralogs indicate that functional diversification is driving the observed increase in gene copy numbers. The induction of prs6 expression in C. rosea during confrontation with F. graminearum suggests an involvement of the corresponding protease in fungal-fungal interactions. The results pinpoint the importance of serine proteases for ecological niche adaptation in C. rosea, including a potential role in the mycoparasitic attack on fungal prey.
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| 9. |
- Iqbal, Mudassir, et al.
(author)
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Deletion of the Nonribosomal Peptide Synthetase Gene nps1 in the Fungus Clonostachys rosea Attenuates Antagonism and Biocontrol of Plant Pathogenic Fusarium and Nematodes
- 2019
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In: Phytopathology. - 0031-949X .- 1943-7684. ; 109, s. 1698-1709
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Journal article (peer-reviewed)abstract
- Secondary metabolites produced by biological control agents may influence the outcome of their interactions with plant pathogenic microorganisms and plants. In the present study, we investigated the role of the nonribosomal peptide synthetase gene nps1 expressed by the biocontrol fungus Clonostachys rosea. A gene expression analysis showed that nps1 was induced during confrontations with the plant pathogenic fungus Botrytis cinerea. Gene deletion strains of nps1 displayed increased growth rates and conidiation. However, the nematicidal activity of culture filtrates from C. rosea Delta nps1 strains was significantly weaker than that from wild-type filtrates (P <= 0.001); after 24 h of incubation with culture filtrates from nps1 deletion strains, only 13 to 33% of a mixed community of nematodes were dead compared with 42% of nematodes incubated with wild-type culture filtrates. The Delta nps1 strains also showed reduced biocontrol efficacy during pot experiments, thus failing to protect wheat seedlings from foot rot disease caused by the plant pathogenic fungus Fusarium graminearum. Furthermore, C. rosea Delta nps1 strains were not able to reduce populations of plant-parasitic nematodes in soil or in roots of wheat as efficiently as the wild-type strain. Both C. rosea wild-type and Delta nps1 strains increased the dry shoot weight and shoot length of wheat by 20 and 13%, respectively. We showed that NPS1, a putative nonribosomal peptide synthetase encoded by nps1, is a biocontrol factor, presumably by producing a hitherto unknown nonribosomal peptide compound with antifungal and nematicidal properties that contributes to the biocontrol properties of C. rosea.
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| 10. |
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