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Soluble material secreted from Penicillium chrysogenum isolate exhibits antifungal activity against Cryphonectria parasitica- the causative agent of the American Chestnut Blight.

ABSTRACT: The American chestnut (Castanea dentata) was once the dominant canopy tree along the eastern region of the United States. Cryphonectria parasitica, the causative agent of chestnut blight, was introduced from Asia in the early 1900's, and obliterated the chestnut population within 50 years. We sought to identify environmental microbes capable of producing factors that were fungicidal or inhibited growth of C. parasitica in the hopes developing a biological control of chestnut blight. We isolated a filamentous fungus that significantly inhibited the growth of C. parasitica upon co-cultivation. Extracellular fractions of this fungal isolate prevented C. parasitica growth, indicating that a potential fungicide was produced by the novel isolate. Sequence analysis of 18S rRNA identified this inhibitory fungus as Penicillium chrysogenum. Furthermore, these extracellular fractions were tested as treatments for blight in vivo using chestnut saplings. Scarred saplings that were treated with the P. chrysogenum extracellular fractions healed subjectively better than those without treatment when inoculated with C. parasitica. These data suggest that material secreted by P. chrysogenum could be used as a treatment for the American chestnut blight. This work may assist the reclamation of the American chestnut in association with breeding programs and blight attenuation. Specifically, treatment of small groves under the right conditions may allow them to remain blight free. Future work will explore the mechanism of action and specific target of the extracellular fraction.

SUBMITTER: Florjanczyk A

PROVIDER: S-EPMC4890969 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Soluble material secreted from Penicillium chrysogenum isolate exhibits antifungal activity against Cryphonectria parasitica- the causative agent of the American Chestnut Blight.

Florjanczyk Aleksandr A Barnes Rebecca R Kenney Adam A Horzempa Joseph J

Journal of plant pathology & microbiology 20160430 4

The American chestnut (Castanea dentata) was once the dominant canopy tree along the eastern region of the United States. Cryphonectria parasitica, the causative agent of chestnut blight, was introduced from Asia in the early 1900's, and obliterated the chestnut population within 50 years. We sought to identify environmental microbes capable of producing factors that were fungicidal or inhibited growth of C. parasitica in the hopes developing a biological control of chestnut ...[more]

PMID: 27274909

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Project description:The ascomycete Cryphonectria parasitica causes destructive chestnut blight. Biological control of the fungus by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe. To provide biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in chestnut trees. Field studies using living trees to date have focused on a selected family of viruses called hypoviruses, especially prototypic hypovirus CHV1, but there are now known to be many other viruses that infect C. parasitica Here, we tested seven different viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in trees. The test included cytosolically and mitochondrially replicating viruses with positive-sense single-stranded RNA or double-stranded RNA genomes. The seven viruses showed different in planta behaviors and were classified into four groups. Group I, including CHV1, had great biocontrol potential and could protect trees by efficiently spreading and converting virulent to hypovirulent cankers in the trees. Group II could induce high levels of hypovirulence but showed much smaller biocontrol potential, likely because of inefficient virus transmission. Group III showed poor performance in hypovirulence induction and biocontrol, while efficiently being transmitted in the infected trees. Group IV could induce hypovirulence and spread efficiently but showed poor biocontrol potential. Nuclear and mitochondrial genotyping of fungal isolates obtained from the treated cankers confirmed virus transmission between the two fungal strains in most isolates. These results are discussed in view of dynamic interactions in the tripartite pathosystem.IMPORTANCE The ascomycete Cryphonectria parasitica causes destructive chestnut blight, which is controllable by hypovirulence-conferring viruses infecting the fungus. The tripartite chestnut/C. parasitica/virus pathosystem involves the dynamic interactions of their genetic elements, i.e., virus transmission and lateral transfer of nuclear and mitochondrial genomes between fungal strains via anastomosis occurring in trees. Here, we tested diverse RNA viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in live chestnut trees. The tested viruses, which are different in genome type (single-stranded or double-stranded RNA) and organization, replication site (cytosol or mitochondria), virus form (encapsidated or capsidless) and/or symptomatology, have been unexplored in the aforementioned aspects under controlled conditions. This study showed intriguing different in-tree behaviors of the seven viruses and suggested that to exert significant biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in the fungus infecting the chestnut trees.

Project description:Plant and fungal interactions drive successional trajectories within reforestation offering both mutualisms (ectomycorrhizal fungi [ECM]) and fungal pathogens. Appalachian forest and mine reclamation projects re-introducing American chestnut and chestnut hybrids will inevitably document the return of chestnut blight, resulting in cankers causing branch dieback and loss of photosynthetic tissue. Similar to herbivory, the loss of photosynthetic tissue may reduce ECM root colonization and cause changes in fungal species composition. To test this, 75 six-year-old established chestnut trees were selected to represent the following: (1) Healthy trees free of chestnut blight; (2) trees with cankers and 50% branch dieback; (3) trees that died prior to the fifth growing season. Each tree had a chestnut seed planted 24 cm from the base. ECM colonization of both the established parent trees (n = 50) and five-month-old seedlings (n = 64) were quantified and genera determined by fungal DNA sequencing of the internal transcribed (ITS) region. Healthier seven-year-old chestnuts trees had significantly more ECM roots than those trees infected with chestnut blight cankers. However, disease die-back on chestnut did not have an influence on community composition among the parent trees or the neighboring five month seedlings. Results also demonstrated that five-month-old seedlings neighboring healthy parent trees had greater ECM on roots (P = 0.002), were larger in size (P = 0.04), and had greater survival (P = 0.01). ECM genera such as Cortinarius, Russula and Scleroderma provided tree to seedling inoculation. ECM colonization by Cortinarius spp. resulted in larger chestnut plants and increased nitrogen foliar concentrations on the five month seedlings. It can be hypothesized that blight will aid in diversifying forest stand composition and these early ECM networks will help facilitate the survival of other native hardwoods that recruit into these sites over time.

Project description:A Ran binding protein (RanBP) homolog, CpRbp1, from Cryphonectria parasitica, has been identified as a protein that is affected by hypovirus infection or tannic acid supplementation. In this study, functional analyses of CpRbp1 were performed by constructing a knockout mutant and analyzing the resulting heterokaryon. Transformation-mediated gene replacement resulted in two putative CpRbp1-null mutants and genotype analyses identified these two mutants as heterokaryotic transformants consisting of two types of nuclei, one with the wild-type CpRbp1 allele and another with the CpRbp1-null mutant allele. Although stable mycelial growth of the heterokaryotic transformant was observed on selective medium containing hygromycin B, neither germination nor growth of the resulting conidia, which were single-cell monokaryotic progeny, was observed on the medium. In trans complementation of heterokaryons using a full-length wild-type allele of the CpRbp1 gene resulted in complemented transformants. These transformants sporulated single-cell monokaryotic conidia that were able to grow on media selective for replacing and/or complementing markers. These results clearly indicate that CpRbp1 is an essential gene, and heterokaryons allowed the fungus to maintain lethal CpRbp1-null mutant nuclei. Moreover, in trans complementation of heterokaryons using chimeric structures of the CpRbp1 gene allowed for analysis of its functional domains, which was previously hampered due to the lethality of the gene. In addition, in trans complementation using heterologous RanBP genes from Aspergillus nidulans was successful, suggesting that the function of RanBP is conserved during evolution. Furthermore, in trans complementation allowed for functional analyses of lethal orthologs. This study demonstrates that our fungal heterokaryon system can be applied effectively to determine whether a gene of interest is essential, perform functional analyses of a lethal gene, and analyze corresponding heterologous genes.

Dataset Information

Soluble material secreted from Penicillium chrysogenum isolate exhibits antifungal activity against Cryphonectria parasitica- the causative agent of the American Chestnut Blight.

Publications

Soluble material secreted from <i>Penicillium chrysogenum</i> isolate exhibits antifungal activity against <i>Cryphonectria parasitica</i>- the causative agent of the American Chestnut Blight.

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