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

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 Pseudomonas, actinomycetes, endospore-forming bacteria, fungi from genera Trichoderma, Gliocladium are known as responsible for inhibition of plant pathogens by various mechanisms (1, 2).

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 Fusarium and to assess the antagonistic capacity of Trichoderma strain in order to be selected as performant bioinoculants.

Soil location: 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%, N^t-0.125%, N-NO₃-7 mg x kg^-1, P_AL-15 mg x kg^-1, K_AL-205 mg x kg^-1).

Microbiological analysis 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 (3).

Fungi identified according to Domsch and Gams (4) and Watanabe (5) 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 (6).

According to the Methodology of microbiological analysis utilized in Romanian soil monitoring system (6), values obtained were interpreted following the criteria:

Total bacteria counts

< 10 x 10⁶ viable cells x g^-1 dry soil - low number

10-20 x 10⁶ viable cells x g^-1 dry soil - moderate number

>20 x 10⁶ viable cells x g^-1 dry soil - high number

Total fungal counts

< 50 x 10³ cfus x g^-1 dry soil - low number

50-100 x 10³ cfus x g^-1 dry soil - moderate number

100 - 200 x 103 cfus x g^-1 dry soil - high number

>200 x 10³ cfus x g^-1 dry soil - very high number

Soil respiration

< 30 mg CO₂ x 100g^-1 soil - low

30-80 mg CO₂ x 100g^-1 soil - moderate

>80 mg CO₂ x 100g^-1 soil - high

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 Trichoderma viride with high cellulolytic abilities presented antagonistic activity against phytopathogenic Fusarium strain.

Our results are in concordance with data from research carried out on Trichoderma strains showing antifungal activity against various plant pathogens including Fusarium (8, 9, 10).

Results from literature reported that biocide formulations with antimicrobial metabolic components extracted from Trichoderma viride alone or from combinations with other fungi were efficient in reducing disease incidence and activating defense response of tomato plants against pathogens (11).

As shown in images of optic microscopy captured in interaction zone between the strains of Trichoderma and Fusarium 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 Trichoderma viride overgrew Fusarium, forming haustoria, pustules and feeding with the content of Fusarium hyphae by enzymatic lysis of the walls.

Similar results with hyperparasitism and biochemical antagonism involved were reported for various Trichoderma strains that showed significant inhibitory effect against fungal pathogenic species (12, 13).

As in our previous research (14) with Trichoderma viride strain SP456 that inhibited Botrytis cinerea, literature cites research with biopreparations of Trichoderma strains (e.g. Trichoderma harzianum-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 (15).

Results of the present work evidenced the antifungal activity of Trichoderma viride 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 Fusarium.

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.

Trichoderma viride presented cellulolytic and antagonistic activity against Fusarium 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 Trichoderma developed its own structures around the hyphae of pathogen Fusarium, emitted haustoria inside to feed and formed pustules.

Results of in vitro assays recommend the Trichoderma viride strain for biotechnological purposes, as antifungal agent in soil inoculation with microbial consortia for the transfer of suppressiveness against pathogen Fusarium.