Protoplast production and regeneration from mycorrhizal fungi and their use for isolation of mutants

1988 ◽  
Vol 34 (2) ◽  
pp. 157-161 ◽  
Author(s):  
Michel Hébraud ◽  
Michel Fèvre
Keyword(s):  
Cell Fusion ◽  
Mycorrhizal Fungi ◽  
Mycorrhizal Fungus ◽  
Lytic Enzymes ◽  
Hebeloma Cylindrosporum ◽  
Regeneration Rate ◽  
Osmotic Stabilizer ◽  
Resistant Mutants ◽  
High Yields ◽  
Protoplast Production

Methods were developed for the reproducible production of high yields of stable protoplasts from monokaryotic and dikaryotic mycelia of Hebeloma cylindrosporum. This is a prerequisite for mutation, cell fusion, and transformation of this mycorrhizal fungus. Protoplasts from Hebeloma cylindrosporum, Hebeloma edurum, Hebeloma sinapizans, and Suillus bellini were released by using cellulase Onozuka R 10 and driselase as lytic enzymes. Production was strongly influenced by the nature and molarity of the osmotic stabilizer and by the mycelial age. In spite of the high percentage of anucleate protoplasts (42%) produced, the regeneration rate averaged 15%. Fungicide-resistant mutants from monokaryotic mycelia of H. cylindrosporum were isolated following ultraviolet irradiation of protoplasts. This indicates that protoplasts will be useful to select physiological mutants from nonsporulating mycelia and to create new strains by mating complementary mycelia or by interspecific cell fusion.

scholarly journals Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

2010 ◽  
Vol 34 (1) ◽  
pp. 74-79 ◽  
Author(s):  
Francine Hiromi Ishikawa ◽  
Quélen de Lima Barcelos ◽  
Elaine Aparecida de Souza ◽  
Eustáquio Souza Dias
Keyword(s):  
Efficient Method ◽  
Incubation Time ◽  
Genetic Manipulation ◽  
Protoplast Isolation ◽  
Colletotrichum Lindemuthianum ◽  
Lytic Enzymes ◽  
Factors Affecting ◽  
Osmotic Stabilizer ◽  
Digestion Mixture ◽  
Protoplast Production

The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.

scholarly journals The methylome of the model arbuscular mycorrhizal fungus, Rhizophagus irregularis, shares characteristics with early diverging fungi and Dikarya

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Anurag Chaturvedi ◽  
Joaquim Cruz Corella ◽  
Chanz Robbins ◽  
Anita Loha ◽  
Laure Menin ◽  
...  
Keyword(s):  
Arbuscular Mycorrhizal Fungi ◽  
Plant Species ◽  
Mycorrhizal Fungi ◽  
Arbuscular Mycorrhizal Fungus ◽  
Mycorrhizal Fungus ◽  
Arbuscular Mycorrhizal ◽  
Rhizophagus Irregularis ◽  
Functional Differences ◽  
Phosphate Regulation ◽  
Very High

AbstractEarly-diverging fungi (EDF) are distinct from Dikarya and other eukaryotes, exhibiting high N6-methyldeoxyadenine (6mA) contents, rather than 5-methylcytosine (5mC). As plants transitioned to land the EDF sub-phylum, arbuscular mycorrhizal fungi (AMF; Glomeromycotina) evolved a symbiotic lifestyle with 80% of plant species worldwide. Here we show that these fungi exhibit 5mC and 6mA methylation characteristics that jointly set them apart from other fungi. The model AMF, R. irregularis, evolved very high levels of 5mC and greatly reduced levels of 6mA. However, unlike the Dikarya, 6mA in AMF occurs at symmetrical ApT motifs in genes and is associated with their transcription. 6mA is heterogeneously distributed among nuclei in these coenocytic fungi suggesting functional differences among nuclei. While far fewer genes are regulated by 6mA in the AMF genome than in EDF, most strikingly, 6mA methylation has been specifically retained in genes implicated in components of phosphate regulation; the quintessential hallmark defining this globally important symbiosis.

scholarly journals Combination of arbuscular mycorrhizal fungi and phosphate solubilizing bacteria on growth and production of Helianthus tuberosus under field condition

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sabaiporn Nacoon ◽  
Sanun Jogloy ◽  
Nuntavun Riddech ◽  
Wiyada Mongkolthanaruk ◽  
Jindarat Ekprasert ◽  
...  
Keyword(s):  
Field Condition ◽  
Mycorrhizal Fungi ◽  
Mycorrhizal Fungus ◽  
Arbuscular Mycorrhizal ◽  
Growth And Yield ◽  
Spore Density ◽  
Phosphate Solubilizing Bacteria ◽  
Phosphate Solubilizing ◽  
Inoculation Treatment ◽  
Amf Inoculation

AbstractIn this work, the effects of co-inoculation between an arbuscular mycorrhizal fungus (AMF) and a phosphate solubilizing bacteria (PSB) to promote the growth and production of sunchoke under field condition were investigated during 2016 and 2017. Four treatments were set up as follows: plants without inoculation, with AMF inoculation, with PSB inoculation and with co-inoculation of PSB and AMF. The results showed the presence of PSB and AMF colonization at the harvest stage in both years. This suggested the survival of PSB and successful AMF colonization throughout the experiments. According to correlation analysis, PSB positively affected AMF spore density and colonization rate. Also, both AMF and PSB positively correlated with growth and production of sunchoke. Co-inoculation could enhance various plant parameters. However, better results in 2016 were found in co-inoculation treatment, while AMF inoculation performed the best in 2017. All of these results suggested that our AMF and PSB could effectively promote growth and production of sunchoke under field conditions. Such effects were varied due to different environmental conditions each year. Note that this is the first study showing successful co-inoculation of AMF and PSB for promoting growth and yield of sunchoke in the real cultivation fields.

Susceptibility of barley cultivars to vesicular-arbuscular mycorrhizal fungi

1995 ◽  
Vol 75 (1) ◽  
pp. 269-275 ◽  
Author(s):  
S. M. Boyetchko ◽  
J. P. Tewari
Keyword(s):  
Mycorrhizal Fungi ◽  
Mycorrhizal Fungus ◽  
Arbuscular Mycorrhizal ◽  
Growth And Yield ◽  
Vam Fungi ◽  
University Of Alberta ◽  
Barley Cultivars ◽  
Vesicular Arbuscular ◽  
Glomus Species ◽  
The University

The relative susceptibility of selected barley cultivars produced in western Canada to vesicular-arbuscular mycorrhizal (VAM) fungi under field and greenhouse conditions was evaluated in this study. Cultivars tested under field conditions at the University of Alberta and Lacombe research stations showed no significant differences in VAM colonization of barley roots; colonization was light. Greenhouse trials at the University of Alberta with eight cultivars inoculated with individual mycorrhizal species illustrated significant differences among the barley cultivars in their reactions to Glomus dimorphicum, G. intraradices, and G. mosseae. Distinct differences were observed in the ability of each Glomus species to colonize the barley cultivars. The VAM fungi increased growth and yield in some cultivars, depending on the Glomus species. This study indicates that a degree of host-specificity exists in VAM fungi and that the host-mycorrhizal fungus genotypes may influence the effectiveness of the symbiosis. Key words: Barley, cultivars, susceptibility, VA mycorrhizal fungi

In vitro culture of arbuscular mycorrhizal fungus and Frankia for inoculation of micropropagated Casuarina equisetifolia L.

1999 ◽  
Vol 77 (9) ◽  
pp. 1391-1397
Author(s):  
Genevieve Louise Mark ◽  
John E Hooker ◽  
Alexander Hahn ◽  
Chris T Wheeler
Keyword(s):  
Spore Germination ◽  
Mycorrhizal Fungi ◽  
Arbuscular Mycorrhizal Fungus ◽  
Mycorrhizal Fungus ◽  
Arbuscular Mycorrhizal ◽  
Dichlorophenoxyacetic Acid ◽  
Casuarina Equisetifolia ◽  
Half Strength ◽  
2,4 Dichlorophenoxyacetic Acid

Micropropagated, rooted, and calli explants of Casuarina equisetifolia L. were inoculated with Frankia UGL 020605S and the arbuscular mycorrhizal fungus (AMF) Glomus mosseae, in single and dual co-culture, in vitro. Different micropropagation media formulations were evaluated for their capacity to stimulate germination of G. mosseae spores and growth of Frankia. Murashige and Skoog basal nutrient (half strength) medium, supplemented with 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D), and pyruvate was selected for the in vitro co-culture of C. equisetifolia callus explants, G. mosseae, and Frankia. This medium (M4) supported 70% AMF spore germination with 44 and 34% of the germinating spores producing single and branched hyphal strands, respectively. Hoaglands (quarter strength, modified by Hoaglands and Arnon (1950)) nutrient medium (M5) with no supplements was selected for the in vitro co-culture of rooted C. equisetifolia explants, G. mosseae, and Frankia and supported 57% AMF spore germination with 29 and 40% of the germinating spores producing single and branched hyphal strands, respectively. Both media supported significant growth of Frankia. In both cases agar was substituted with Terragreen(r). AMF appressoria and intercellular hyphae were observed in rooted C. equisetifolia at 28 days; arbuscule formation occurred at 56 days postinoculation. Frankia infection was evident after 28 days. This was observed in both dual and single in vitro co-cultures. No specific immunofluorescent or immunogold reactions to monoclonal antibodies (mABs) anti-Frankia < 8C5 > and anti-G. mosseae < F5G5 > were evident in C. equisetifolia callus explants.Key words: arbuscular mycorrhizal fungi (AMF), Frankia, Casuarina, micropropagation, immunofluorescent labelling.

Effects of forest litter on mycorrhiza development and growth of Douglas-fir and western red cedar seedlings

1983 ◽  
Vol 13 (4) ◽  
pp. 666-671 ◽  
Author(s):  
Jennifer L. Parke ◽  
R. G. Llnderman ◽  
J. M. Trappe
Keyword(s):  
Organic Matter ◽  
Mycorrhizal Fungi ◽  
Mycorrhizal Fungus ◽  
Forest Litter ◽  
Douglas Fir ◽  
Growth Enhancement ◽  
Inoculum Potential ◽  
Red Cedar ◽  
Western Red Cedar ◽  
Slash Burning

Preparation of forest regeneration sites prior to conifer planting often includes slash burning or physical removal of soil organic matter. Experiments were conducted to determine if organic matter contributes to the mycorrhizal fungus inoculum potential in forest soils and to compare the growth of Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco) and western red cedar (Thujaplicata J. Donn ex D. Donn) seedlings with and without litter. Litter and humus were found to include inoculum of both vesicular–arbuscular (VA) and ectomycorrhizal fungi. Litter amendment usually enhanced growth of host seedlings, but growth enhancement could not be fully attributed to addition of mycorrhizal inoculum or nutrients provided by litter. These findings suggested that other biological factors stimulated the growth of conifer seedlings and (or) activity of mycorrhizal fungi.

scholarly journals Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms

2016 ◽  
Vol 62 (7) ◽  
pp. 579-587 ◽  
Author(s):  
Xiuyan Wei ◽  
Xinyue Song ◽  
Dong Dong ◽  
Nemat O. Keyhani ◽  
Lindan Yao ◽  
...  
Keyword(s):  
Rational Design ◽  
Pathogenic Fungus ◽  
Scale Insects ◽  
Critical Parameters ◽  
Selection Marker ◽  
Efficient Production ◽  
Design Strategies ◽  
Osmotic Stabilizer ◽  
Optimized Protocol ◽  
Protoplast Production

The insect pathogenic fungus Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. However, few genetic tools are currently available for studying this organism. Here we report on the conditions for the production of transformable A. placenta protoplasts using an optimized protocol based on the response surface method (RSM). Critical parameters for protoplast production were modelled by using a Box–Behnken design (BBD) involving 3 levels of 3 variables that was subsequently tested to verify its ability to predict protoplast production (R2 = 0.9465). The optimized conditions resulted in the highest yield of protoplasts ((4.41 ± 0.02) × 107 cells/mL of culture, mean ± SE) when fungal cells were treated with 26.1 mg/mL of lywallzyme for 4 h of digestion, and subsequently allowed to recover for 64.6 h in 0.7 mol/L NaCl–Tris buffer. The latter was used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the nonoptimized conditions. Generated protoplasts were transformed with vector PbarGPE containing the bar gene as the selection marker. Transformation efficiency was 300 colonies/(μg DNA·107 protoplasts), and integration of the vector DNA was confirmed by PCR. The results show that rational design strategies (RSM and BBD methods) are useful to increase the production of fungal protoplasts for a variety of downstream applications.

scholarly journals Towards Growth of Arbuscular Mycorrhizal Fungi Independent of a Plant Host

2002 ◽  
Vol 68 (4) ◽  
pp. 1919-1924 ◽  
Author(s):  
Ulrich Hildebrandt ◽  
Katharina Janetta ◽  
Hermann Bothe
Keyword(s):  
Mycorrhizal Fungi ◽  
Glomus Intraradices ◽  
Mycorrhizal Fungus ◽  
Fungal Growth ◽  
Arbuscular Mycorrhizal ◽  
Plant Host ◽  
Significant Step ◽  
Hyphal Coils ◽  
K 12 ◽  
Root Tissues

ABSTRACT When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Sy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G. Bécard and J. A. Fortin (New Phytol. 108:211-218, 1988), slime-forming bacteria, identified as Paenibacillus validus, frequently grew up. These bacteria were able to support growth of the fungus on the agar plates. In the presence of P. validus, hyphae branched profusely and formed coiled structures. These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA from Agrobacterium rhizogenes. The presence of P. validus alone also enabled G. intraradices to form new spores, mainly at the densely packed hyphal coils. The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei. In these experiments P. validus could not be replaced by bacteria such as Escherichia coli K-12 or Azospirillum brasilense Sp7. Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) from P. validus which stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.

Use of lytic enzymes for protoplast production in Trichoderma reesei QM9414

1989 ◽  
Vol 11 (1) ◽  
pp. 21-25 ◽  
Author(s):  
Dhanwant K. Sandhu ◽  
Vipul Wadhwa ◽  
Paramjeet S. Bagga
Keyword(s):  
Trichoderma Reesei ◽  
Lytic Enzymes ◽  
Protoplast Production ◽  
Trichoderma Reesei Qm9414

Tripartite associations in snowbrush (Ceanothus velutinus): effect of vesicular–arbuscular mycorrhizae on growth, nodulation, and nitrogen fixation

1981 ◽  
Vol 59 (1) ◽  
pp. 34-39 ◽  
Author(s):  
S. L. Rose ◽  
C. T. Youngberg
Keyword(s):  
Host Plant ◽  
Mycorrhizal Fungi ◽  
Arbuscular Mycorrhizae ◽  
Nitrogenase Activity ◽  
Mycorrhizal Fungus ◽  
Nodule Formation ◽  
Nitrogen Fixing ◽  
Symbiotic Associations ◽  
Vesicular Arbuscular ◽  
Va Mycorrhizal Fungi

Symbiotic associations were established between nitrogen-fixing nonleguminous (actinorrhizal) snowbrush (Ceanothus velutinus Dougl.) seedlings and two categories of microorganisms: vesicular–arbuscular (VA) mycorrhizal fungi and a filamentous actinomycete capable of inducing nodule formation. The actinomycete is housed in nodules where fixation of atmospheric dinitrogen occurs and is made available to the host plant; the mycorrhizal fungus is both inter- and intra-cellular within the root tissue and may be found within the nodules. The two major nutrients, N and P, are made available and can be supplied to the host plant by these two symbiotic microorganisms. The root system of snowbrush seedlings was dually colonized by VA mycorrhizal fungi and a nitrogen-fixing actinomycete and the possibility of a direct interaction between the endophytes in the symbioses was investigated. Dually infected plants showed increases in total dry weight of shoots and roots, number of nodules, weight of nodular tissue, as well as higher levels of N, Ca2+, and P, and an increase in nitrogenase activity as measured by acetylene reduction.

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