<abstract xmlns="http://www.w3.org/1999/xhtml">Soils from rural zones with high natural value (HNV) agriculture systems are an important source of beneficial microbial species that can be useful for various biotechnological purposes, such as transfer of suppressiveness against plant pathogens from suppressive to disease-inducing soils by using inoculation with antagonistic selected strains.The main goal of the paper was to present the results of the research carried out on strains isolated from soil microbial populations in HNV agriculture system (Mureș county, Romania) responsible for specific suppressiveness against soil-borne phytopathogens. The dual culture method was used for assessing the mechanisms involved in antagonism against a plant pathogenic strain from genus <italic>Fusarium</italic>.The global microbial activity measured as soil respiration was intense. Total counts of bacteria and fungi estimated by dilution plate were also high.The community of heterotrophic aerobic bacteria included 13 species. Associations of fluorescent pseudomonads and actinomycetes were dominant and presented antagonistic activity against <italic>Fusarium</italic>.Twenty fungal species presented cellulolytic capability evidenced by growth on culture media with cellulose as sole source of carbon. Over cellulolytic capacity, the selected isolate of Trichoderma viride presented antagonistic activity against pathogenic <italic>Fusarium</italic> strain. Both biochemical mechanism and hyperparasitism were evidenced as involved in its antifungal activity.</abstract>

Soils from rural zones with high natural value (HNV) agriculture systems are an important source of beneficial microbial species that can be useful for various biotechnological purposes, such as transfer of suppressiveness against plant pathogens from suppressive to disease-inducing soils by using inoculation with antagonistic selected strains.The main goal of the paper was to present the results of the research carried out on strains isolated from soil microbial populations in HNV agriculture system (Mureș county, Romania) responsible for specific suppressiveness against soil-borne phytopathogens. The dual culture method was used for assessing the mechanisms involved in antagonism against a plant pathogenic strain from genus Fusarium.The global microbial activity measured as soil respiration was intense. Total counts of bacteria and fungi estimated by dilution plate were also high.The community of heterotrophic aerobic bacteria included 13 species. Associations of fluorescent pseudomonads and actinomycetes were dominant and presented antagonistic activity against Fusarium.Twenty fungal species presented cellulolytic capability evidenced by growth on culture media with cellulose as sole source of carbon. Over cellulolytic capacity, the selected isolate of Trichoderma viride presented antagonistic activity against pathogenic Fusarium strain. Both biochemical mechanism and hyperparasitism were evidenced as involved in its antifungal activity.

<div xmlns="http://www.w3.org/1999/xhtml"><sec id="j_ebtj-2018-0049_s_001_w2aab3b7b2b1b6b1ab1aAa"><div>Introduction</div>Agriculture of high natural value (HNV- High Natural Value) represents a recent (last two decades) concept describing those agricultural systems with practices allowing preservation of high biodiversity in Europe. This concept recognizes that preservation of biodiversity depends on traditional agricultural practices but also on applying specific agricultural practices in large zones of rural European space.The soils in these HNV systems are an important source of beneficial microbial species that can be useful for various biotechnological purposes, such as transfer of suppressiveness against plant pathogens from suppressive to disease-inducing soils by using inoculation with specific selected strains.Fluorescent and non-fluorescent species of <italic>Pseudomonas</italic>, actinomycetes, endospore-forming bacteria, fungi from genera <italic>Trichoderma</italic>, <italic>Gliocladium</italic> are known as responsible for inhibition of plant pathogens by various mechanisms (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_001_w2aab3b7b2b1b6b1ab1b7b1Aa">1</a>, <a ref-type="bibr" href="#j_ebtj-2018-0049_ref_002_w2aab3b7b2b1b6b1ab1b7b2Aa">2</a>).The goal of the present paper was to present the results of the research carried out on bacteria and fungi isolated from soil microbial populations in high natural value agriculture system (Mureș county, Romania) responsible for specific suppressiveness against soil-borne phytopathogen <italic>Fusarium</italic> and to assess the antagonistic capacity of <italic>Trichoderma</italic> strain in order to be selected as performant bioinoculants.</sec><sec id="j_ebtj-2018-0049_s_002_w2aab3b7b2b1b6b1ab1b1Aa"><div>Materials and Methods</div><bold><italic>Soil location</italic></bold>: Soil samples (0-20cm) collected from high natural value (HNV) agriculture system, habitat 6210, Pilot area 1, Apold, Mureș county, belonging to European network of protected areas, Nature 2000(NGO property), 524 m altitude, 460 8’4” N latitude and 240 49’ 16” E longitude, under grassland without anthropic intervention (pH-8.39, humus-4.02%, Nt-0.125%, N-NO3-7 mg x kg-1, PAL-15 mg x kg-1, KAL-205 mg x kg-1).<bold><italic>Microbiological analysis</italic></bold> of soil sampled in September 2017, after hay harvesting from high natural value farming system was performed by plating soil decimal dilutions on specific solid culture media, Topping for heterotrophic aerobic bacteria, Czapek for fungi and Stapp for cellulolytic microorganisms.After 7 days incubation at dark, microbial colonies developed were counted and their density was reported to gram of dry soil.Taxonomic identification of bacteria was done according to Bergey’s manual (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_003_w2aab3b7b2b1b6b1ab1b7b3Aa">3</a>).Fungi identified according to Domsch and Gams (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_004_w2aab3b7b2b1b6b1ab1b7b4Aa">4</a>) and Watanabe (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_005_w2aab3b7b2b1b6b1ab1b7b5Aa">5</a>) determinative manuals on the basis of colony morphology and structural characteristics observed by optic microscopy.The global physiological activities of microflora were determined by substrate induced respiration method (SIR) and expressed as mg CO2 x100g-1 soil (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_006_w2aab3b7b2b1b6b1ab1b7b6Aa">6</a>).According to the Methodology of microbiological analysis utilized in Romanian soil monitoring system (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_006_w2aab3b7b2b1b6b1ab1b7b6Aa">6</a>), values obtained were interpreted following the criteria:<italic>Total bacteria counts</italic>&lt; 10 x 106 viable cells x g-1 dry soil - low number10-20 x 106 viable cells x g-1 dry soil - moderate number>20 x 106 viable cells x g-1 dry soil - high number<italic>Total fungal counts</italic>&lt; 50 x 103 cfus x g-1 dry soil - low number50-100 x 103 cfus x g-1 dry soil - moderate number100 - 200 x 103 cfus x g-1 dry soil - high number>200 x 103 cfus x g-1 dry soil - very high number<italic>Soil respiration</italic>&lt; 30 mg CO2 x 100g-1 soil - low30-80 mg CO2 x 100g-1 soil - moderate>80 mg CO2 x 100g-1 soil - high<sec id="j_ebtj-2018-0049_s_002_s_001_w2aab3b7b2b1b6b1ab1b1c21Aa"><div>Antagonistic activity of microbial strains against <italic>Fusarium</italic> pathogen</div>The microbial antagonistic capacity and mechanisms involved were assessed by dual culture method (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_007_w2aab3b7b2b1b6b1ab1b7b7Aa">7</a>) against soil-borne plant pathogenic strain from genus <italic>Fusarium</italic>. Colonies were monitored for morphological characteristics, sporulation, production of metabolites and the presence of interaction zone.Micrographs were done to reveal by optical microscopy the aspects of antagonism and its nature.</sec></sec><sec id="j_ebtj-2018-0049_s_003_w2aab3b7b2b1b6b1ab1b2Aa"><div>Results</div><sec id="j_ebtj-2018-0049_s_003_s_001_w2aab3b7b2b1b6b1ab1b2b1Aa"><div>Microbiological analysis</div><sec id="j_ebtj-2018-0049_s_003_s_001_s_001_w2aab3b7b2b1b6b1ab1b2b1b1Aa"><div>Quantitative assays (total counts and global microbial activities)</div>Total microbial activity measured as soil respiration was 82.590 mg CO2 x 100g-1 soil.The total counts estimated by dilution plate method were 38.815 x 106 viable cells x g-1 dry soil for heterotrophic aerobic bacteria and 179.968 x 103 colony forming units x g-1 dry soil for microscopic fungi.<table-wrap id="j_ebtj-2018-0049_tab_001_w2aab3b7b2b1b6b1ab1b2b1b1b3Aa" position="float"><label>Table 1</label><caption>Taxonomic composition of bacterial and fungal microflora in soil from Apold</caption><table rules="groups"><thead><tr><th align="left">Soil profile</th><th align="left">Bacterial species</th><th align="left">Fungal species</th></tr></thead><tbody><tr><td align="left"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_ebtj-2018-0049_ingr_001.png"></inline-graphic></td><td align="left"><italic>Pseudomonas fluorescens</italic>,</td><td align="left"><italic>Humicola grisea</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Bacillus megaterium</italic></td><td align="left"><italic>Penicillium glabrum</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Bacillus cereus</italic> var. <italic>mycoides</italic></td><td align="left"><italic>Trichoderma viride</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Bacillus cereus</italic></td><td align="left"><italic>Penicillium janthinellum</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Pseudomonas acidophila</italic></td><td align="left"><italic>Paecilomyces marquandii</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Bacillus</italic> sp.</td><td align="left"><italic>Cladosporium herbarum</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Pseudomonas</italic> sp.</td><td align="left"><italic>Absidia spinosa</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Pseudomonas pseudogleyi</italic></td><td align="left"><italic>Aspergillus terreus</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Bacillus circulans Serratia marcescens</italic></td><td align="left"><italic>Mucor vesiculosus Fusarium oxysporum</italic></td></tr><tr><td align="left"></td><td align="left"><italic>Bacillus subtilis</italic></td><td align="left"><italic>Aspergillus niger</italic></td></tr><tr><td align="left"></td><td align="left">Actinomycetes Series Albus and</td><td align="left"><italic>Verticillium</italic> sp.</td></tr><tr><td align="left"></td><td align="left">Griseus</td><td align="left"><italic>Aureobasidium pullulans</italic></td></tr><tr><td align="left"></td><td align="left"></td><td align="left"><italic>Cunninghamella elegans</italic></td></tr><tr><td align="left"></td><td align="left"></td><td align="left"><italic>Cladosporium sphaerospermum</italic></td></tr><tr><td align="left"></td><td align="left"></td><td align="left"><italic>Stachybotrys chartarum</italic></td></tr><tr><td align="left"></td><td align="left"></td><td align="left"><italic>Penicillium</italic> sp.</td></tr><tr><td align="left"></td><td align="left"></td><td align="left"><italic>Penicillium aurantiogriseum</italic></td></tr><tr><td align="left"></td><td align="left"></td><td align="left"><italic>Geotrichum candidum</italic></td></tr><tr><td align="left"></td><td align="left"></td><td align="left"><italic>Aspergillus fumigatus</italic></td></tr></tbody></table></table-wrap></sec><sec id="j_ebtj-2018-0049_s_003_s_001_s_002_w2aab3b7b2b1b6b1ab1b2b1b2Aa"><div>Taxonomic composition of soil microbial communities</div>Bacterial community included 13 species, dominated by fluorescent pseudomonads and actinomycetes from series Albus with antagonistic activity against mycotoxigenic strains of <italic>Fusarium</italic>, accompanied by representatives of genera <italic>Bacillus</italic> and <italic>Serratia</italic> (<bold><a ref-type="table" href="#j_ebtj-2018-0049_tab_001_w2aab3b7b2b1b6b1ab1b2b1b1b3Aa">Table 1</a></bold>).A number of 20 fungal species were identified (<bold><a ref-type="table" href="#j_ebtj-2018-0049_tab_001_w2aab3b7b2b1b6b1ab1b2b1b1b3Aa">Table 1</a></bold>), known for their large adaptability and metabolic ability to decompose various substrates.Many strains presented the ability to grow on Stapp culture media with cellulose as sole source of carbon (e.g. from genera <italic>Stachybotrys, Aspergillus, Cladosporium, Cunninghamella, Paecilomyces, Penicillium, Humicola</italic>).</sec></sec><sec id="j_ebtj-2018-0049_s_003_s_002_w2aab3b7b2b1b6b1ab1b2b2Aa"><div>Antagonistic activity of microbial strain against Fusarium pathogen</div>Over cellulolytic capacity (<bold><a ref-type="fig" href="#j_ebtj-2018-0049_fig_002_w2aab3b7b2b1b6b1ab1b2b2b3Aa">Fig. 2</a></bold>), the isolate of <italic>Trichoderma viride</italic> presented antagonistic activity against <italic>Fusarium</italic> strain with both biochemical mechanism and hyperparasitism involved, as evidenced by macroscopic and optic microscopy examination of interaction zone (<bold><a ref-type="fig" href="#j_ebtj-2018-0049_fig_001_w2aab3b7b2b1b6b1ab1b2b2b2Aa">Fig. 1</a></bold> and <bold><a ref-type="fig" href="#j_ebtj-2018-0049_fig_003_w2aab3b7b2b1b6b1ab1b2b2b4Aa">Fig. 3</a></bold>).<figure id="j_ebtj-2018-0049_fig_001_w2aab3b7b2b1b6b1ab1b2b2b2Aa" position="float" fig-type="figure"><h2>Figure 1</h2><figCaption>Interaction zone with <italic>Fusarium</italic> overgrown by <italic>Trichoderma viride</italic>.</figCaption><img xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_ebtj-2018-0049_fig_001.jpg" src="https://sciendo-parsed.s3.eu-central-1.amazonaws.com/6471b835215d2f6c89dae8f2/j_ebtj-2018-0049_fig_001.jpg?X-Amz-Algorithm=AWS4-HMAC-SHA256&amp;X-Amz-Date=20240603T073349Z&amp;X-Amz-SignedHeaders=host&amp;X-Amz-Expires=18000&amp;X-Amz-Credential=AKIA6AP2G7AKP25APDM2%2F20240603%2Feu-central-1%2Fs3%2Faws4_request&amp;X-Amz-Signature=8d277920976c0d2054704769b60b52d340745417a6a46ab821b98d52bb934a68" class="mw-100"/></figure><figure id="j_ebtj-2018-0049_fig_002_w2aab3b7b2b1b6b1ab1b2b2b3Aa" position="float" fig-type="figure"><h2>Figure 2</h2><figCaption>The cellulolytic capacity of <italic>Trichoderma viride</italic> evidenced by growth on Stapp medium with filter paper as sole source of carbon.</figCaption><img xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_ebtj-2018-0049_fig_002.jpg" src="https://sciendo-parsed.s3.eu-central-1.amazonaws.com/6471b835215d2f6c89dae8f2/j_ebtj-2018-0049_fig_002.jpg?X-Amz-Algorithm=AWS4-HMAC-SHA256&amp;X-Amz-Date=20240603T073349Z&amp;X-Amz-SignedHeaders=host&amp;X-Amz-Expires=18000&amp;X-Amz-Credential=AKIA6AP2G7AKP25APDM2%2F20240603%2Feu-central-1%2Fs3%2Faws4_request&amp;X-Amz-Signature=2f0fa5dc273ee9da00deb6efe13e9077658d90fcc832607a41e2a277e54de4fe" class="mw-100"/></figure><figure id="j_ebtj-2018-0049_fig_003_w2aab3b7b2b1b6b1ab1b2b2b4Aa" position="float" fig-type="figure"><h2>Figure 3</h2><figCaption>Hyphae and haustoria of <italic>Trichoderma viride</italic> hyperparasite on <italic>Fusarium</italic> hyphae.</figCaption><img xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_ebtj-2018-0049_fig_003.jpg" src="https://sciendo-parsed.s3.eu-central-1.amazonaws.com/6471b835215d2f6c89dae8f2/j_ebtj-2018-0049_fig_003.jpg?X-Amz-Algorithm=AWS4-HMAC-SHA256&amp;X-Amz-Date=20240603T073349Z&amp;X-Amz-SignedHeaders=host&amp;X-Amz-Expires=18000&amp;X-Amz-Credential=AKIA6AP2G7AKP25APDM2%2F20240603%2Feu-central-1%2Fs3%2Faws4_request&amp;X-Amz-Signature=0bae153365a7040e3b0ef3357188804701b0aaa5502d0a8344284bfd6437a077" class="mw-100"/></figure>Morphology and sporulation pattern of phytopathogen were modified (development and sporulation delays, hyphal twisting).Hyperparasitism was evidenced when <italic>Trichoderma</italic> developed its own structures around the hyphae of pathogen, emitted haustoria inside it to feed and formed pustules that overgrew <italic>Fusarium</italic> colony.Biochemical interaction was revealed by the existence of yellow metabolites more intensely released in the inhibition zone, well visible on the back side of the Petri dish.</sec></sec><sec id="j_ebtj-2018-0049_s_004_w2aab3b7b2b1b6b1ab1b3Aa"><div>Discussion</div>The results of the research carried out demonstrated the existence of microbial species diversity in soil from HNV agriculture system (Apold, Mureș county), including thirteen bacterial and twenty fungal taxa. The isolate of <italic>Trichoderma viride</italic> with high cellulolytic abilities presented antagonistic activity against phytopathogenic <italic>Fusarium</italic> strain.Our results are in concordance with data from research carried out on <italic>Trichoderma</italic> strains showing antifungal activity against various plant pathogens including <italic>Fusarium</italic> (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_008_w2aab3b7b2b1b6b1ab1b7b8Aa">8</a>, <a ref-type="bibr" href="#j_ebtj-2018-0049_ref_009_w2aab3b7b2b1b6b1ab1b7b9Aa">9</a>, <a ref-type="bibr" href="#j_ebtj-2018-0049_ref_010_w2aab3b7b2b1b6b1ab1b7c10Aa">10</a>).Results from literature reported that biocide formulations with antimicrobial metabolic components extracted from <italic>Trichoderma viride</italic> alone or from combinations with other fungi were efficient in reducing disease incidence and activating defense response of tomato plants against pathogens (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_011_w2aab3b7b2b1b6b1ab1b7c11Aa">11</a>).As shown in images of optic microscopy captured in interaction zone between the strains of <italic>Trichoderma</italic> and <italic>Fusarium</italic> from dual culture, the metabolites of antagonist visible on the reverse of Petri plate suppressed the growth and fructification of pathogen.Later, the colony of <italic>Trichoderma viride</italic> overgrew <italic>Fusarium</italic>, forming haustoria, pustules and feeding with the content of <italic>Fusarium</italic> hyphae by enzymatic lysis of the walls.Similar results with hyperparasitism and biochemical antagonism involved were reported for various <italic>Trichoderma</italic> strains that showed significant inhibitory effect against fungal pathogenic species (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_012_w2aab3b7b2b1b6b1ab1b7c12Aa">12</a>, <a ref-type="bibr" href="#j_ebtj-2018-0049_ref_013_w2aab3b7b2b1b6b1ab1b7c13Aa">13</a>).As in our previous research (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_014_w2aab3b7b2b1b6b1ab1b7c14Aa">14</a>) with <italic>Trichoderma viride</italic> strain SP456 that inhibited <italic>Botrytis cinerea</italic>, literature cites research with biopreparations of <italic>Trichoderma</italic> strains (e.g. <italic>Trichoderma harzianum</italic>-T22, well-known under commercial brand Root Shield) for the biocontrol of pathogenic fungi and explain their inhibitory properties by the ability to produce various enzymes and metabolites from the group of trichothecenes with antifungal properties (<a ref-type="bibr" href="#j_ebtj-2018-0049_ref_015_w2aab3b7b2b1b6b1ab1b7c15Aa">15</a>).Results of the present work evidenced the antifungal activity of <italic>Trichoderma viride</italic> isolated from soil in HNV agriculture system from Apold by biochemical antagonism and hyperparasitism and recommend it for utilization in biotechnological strategies of suppressiveness transfer against phytopathogenic fungi from genus <italic>Fusarium</italic>.</sec><sec id="j_ebtj-2018-0049_s_005_w2aab3b7b2b1b6b1ab1b4Aa"><div>Conclusions</div>The well-developed and active microbial populations from soil in high natural value system under grassland, with 13 bacterial and 20 fungal taxa were dominated by fluorescent pseudomonads, actinomycetes and included many cellulolytic species of fungi.<italic>Trichoderma viride</italic> presented cellulolytic and antagonistic activity against <italic>Fusarium</italic> strain with both biochemical mechanism and hyperparasitism involved.Morphology and sporulation pattern were modified (delays, discoloration) and biochemical interaction was revealed by the existence of yellow metabolites more intensely released in the inhibition zone.Hyperparasitism was evidenced when <italic>Trichoderma</italic> developed its own structures around the hyphae of pathogen <italic>Fusarium</italic>, emitted haustoria inside to feed and formed pustules.Results of <italic>in vitro</italic> assays recommend the <italic>Trichoderma viride</italic> strain for biotechnological purposes, as antifungal agent in soil inoculation with microbial consortia for the transfer of suppressiveness against pathogen <italic>Fusarium</italic>.</sec></div>

Soils from HNV agriculture systems as source of microorganisms with antifungal activity

The EuroBiotech Journal

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Soils from rural zones with high natural value (HNV) agriculture systems are an important source of beneficial microbial species that can be useful for various...

Soils from HNV agriculture systems as source of microorganisms with antifungal activity

Article Category: Research Article

Published Online: Oct 27, 2018

Page range: 196 - 199

DOI: https://doi.org/10.2478/ebtj-2018-0049

Keywords
microorganisms, antagonistic activity, phytopathogenic, soil suppressiveness

© 2018 Matei Sorin, Matei Gabi-Mirela, Dumitrașcu Monica, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Soils from HNV agriculture systems as source of microorganisms with antifungal activity

Article Category: Research Article

Published Online: Oct 27, 2018

Page range: 196 - 199

DOI: https://doi.org/10.2478/ebtj-2018-0049

Keywordsmicroorganisms, antagonistic activity, phytopathogenic, soil suppressiveness

© 2018 Matei Sorin, Matei Gabi-Mirela, Dumitrașcu Monica, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Keywords
microorganisms, antagonistic activity, phytopathogenic, soil suppressiveness