Next Article in Journal
Agricultural Waste Valorization: Exploring Environmentally Friendly Approaches to Bioenergy Conversion
Previous Article in Journal
Optimized Battery Capacity Allocation Method for Wind Farms with Dual Operating Conditions
Previous Article in Special Issue
An Eretmocerus Species, Parasitoid of Aleurocanthus spiniferus, Was Found in Europe: The Secret Savior of Threatened Plants
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Sustainability
Volume 16
Issue 9
10.3390/su16093616
sustainability-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

The Effect of Humic Substances on the Colony Growth and Conidial Germination of Entomopathogenic Fungi from the Genus Metarhizium

by
Anna Majchrowska-Safaryan
1,
Cezary Tkaczuk
1,* and
Marta Wrzosek
2
1
Faculty of Agricultural Sciences, Institute of Agriculture and Horticulture, University of Siedlce, 08-110 Siedlce, Poland
2
Botanical Garden, University of Warsaw, 00-478 Warsaw, Poland
*
Author to whom correspondence should be addressed.
Sustainability 2024, 16(9), 3616; https://0-doi-org.brum.beds.ac.uk/10.3390/su16093616
Submission received: 18 March 2024 / Revised: 19 April 2024 / Accepted: 22 April 2024 / Published: 26 April 2024
(This article belongs to the Special Issue Toward Sustainable Agriculture: Crop Protection and Pest Control)
Browse Figures
Versions Notes

Abstract

:
The development of sustainable agricultural production involves the use of new plant protection products, with low toxicity to non-target organisms and high biodegradability. The aim of this study was to investigate the effect of commercially available preparations containing humic substances, in comparison with pure humic acids, on the growth and germination of spores of entomopathogenic fungi (EPFs) from the genus Metarhizium in vitro. AmiAGRA, HumiAGRA, AlgoHUM (recommended field dose) and pure humic acids extracted from peat, brown coal and spent mushroom substrate were added to Sabouraud’s culture substrate. Observation of the growth of the colonies of the tested species of EPFs was carried out every 5 days until day 20, measuring their diameter (mm). In the second stage of the experiment, the culture medium with the addition of preparations and pure humic acids was applied in a thin layer to the surface of glass slides and an aqueous solution with spores was introduced. Observation was carried out after 24 and 48 h, and the results obtained were expressed as percentages in relation to the control. The conducted research showed that on the 20th day of the culture (on average), preparations containing humic substances had a stimulating effect, while pure acids limited the growth of the colonies of the tested isolates slightly. The growth of M. anisopliae fungal colonies was most strongly stimulated by AlgoHUM, and M. flavoviride by HumiAGRA. The share of germinated spores after 48 h of contact with the substrate was higher than after 24 h, and more spores germinated on substrates with the addition of preparations containing humic substances than on pure humic acids.

1. Introduction

In the European Union countries, the European Green Deal and the Farm to Fork Strategy have been introduced into agricultural practice to minimize the use of chemicals in favor of biological products, including beneficial microorganisms. As a result, numerous bioproducts have been tested and implemented in regard to crop production toward sustainable agriculture [1,2]. They are based on natural biological materials, such as plant extracts, polysaccharides and humic substances. Products containing humic substances stimulate the decomposition of organic matter introduced into the soil and, as a result, the released nutrients become available to plants more quickly [3]. Additionally, humic acids can be used as adjuvants to increase the effectiveness of biocontrol agents [4] and as carriers for microorganisms [5]. Humic substances extracted from lignite or peat are most often applied to lighter soils [6,7]. Humic acids are considered to be biocompatible and environmentally friendly [8,9].
Bioproducts containing beneficial bacteria and fungi are also used to protect plants against pests [10]. Due to their ability to directly penetrate the body of insects through the epidermis, EPFs are relatively highly effective when integrated into pest control strategies. Their conidia adhere to the insect epidermis, germinate and penetrate the host, which eventually dies [11,12,13]. After the death of the insect, hyphae emerge from its corpse and produce conidiophores and conidia, infecting new hosts through horizontal transmission [12]. There has been increasing interest in microbiological products among farmers due to the decreasing number of chemical pesticides approved by the EU [14]. Mycoinsecticides, currently available in Poland, are based on three EPFs: Beauveria bassiana strain ATCC 74040, Metarhizium anisopliae var. anisopliae strain F52 and Isaria fumosorosea (=Cordyceps fumosorosea) strain Apopka 97. The first one has been registered to control wireworms, but only in the cultivation of eggplants, bell peppers and tomatoes grown under cover. In turn, M. anisopliae var. anisopliae strain F52 is used to control vine weevils, a species of beetle, in the cultivation of, among others, currants and strawberries. PreFeRal containing the Apopka 97 strain is used to stop whitefly from attacking plants grown under cover [15,16].
The aim of this study was to determine the effectiveness of commercially available products containing humic substances, in comparison with the effectiveness of pure humic acids, on colony growth and spore germination of EPFs from the genus Metarhizium.

2. Materials and Methods

2.1. Fungal Isolates

The effect of preparations containing humic substances and pure humic acids on EPF colony growth and on spore germination was investigated in laboratory conditions. Two selected species from the Metarhizium genus were used in the research: the fungus M. anisopliae (Metschn.) Sorokin (M05-UPH), isolated from the soil of a cultivated field with Galleria mellonella larvae in Chodów (Mazowieckie Voivodeship, Poland); and M. flavoviride W. Gams and Rozsypal (M06-UPH), isolated from the soil of a cultivated field with Galleria mellonella larvae in Samowicze (Lubelskie Voivodeship, Poland). The cultures of the fungi used in this experiment were deposited in the fungal collection of the Institute of Agriculture and Horticulture, the University of Siedlce, Poland, and stored in an SDA medium at 4 °C. They were identified macroscopically using standard keys [17,18], and their systematic affiliation was also confirmed by molecular identification. The ITS marker, proposed as a universal DNA code marker for fungi, was chosen for identification [19]. Molecular identification of isolates was carried out in the mycological laboratory at the Biological and Chemical Research Centre of the University of Warsaw, using Qiagen and Blirt tools (DNA isolation kits, PCR kit and cleaning kit). The PCR reaction was carried out according to the procedure provided by Kovač et al. [20]. Sanger sequencing was used with single ITS2, ITS3, ITS4 and ITS5 primers and the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Waltham, MA, USA), containing fluorescently labeled dideoxynucleotide triphosphates (ddNTPs), deoxynucleotide triphosphates (dNTPs), Taq-FS polymerase and buffer. After sequencing, product cleaning was conducted by molecular filtration on columns with Sephadex G-50, and the reading of the result was entrusted to Genomed (Warsaw). The sequences were compared to those available in the NCBI database using the BLASTN 2.2.2 algorithm [21].

2.2. Preparations and Pure Humic Acids Used

Preparations containing humic substances including AmiAGRA, HumiAGRA and AlgoHUM (recommended field dose) and humic acids extracted from peat, lignite and spent mushroom substrate (SMS) were used in the experiment. Based on the content of the humic substances in the AmiAGRA preparation, the dose of pure humic acids was calculated. Fractions of organic matter from peat, lignite and SMS were obtained on the basis of sequential fractionation carbon compounds. The sequential fractionation of carbon compounds was performed based on the Schnitzer method. The pure humic acids were calculated on an ash-free mass [22,23,24]. The characteristics of the preparations and pure humic acids used are presented in Table 1.
In the experiment, Sabouraud dextrose agar (SDA) produced by bioMérieux was used as a culture medium, with casein enzymatic hydrolyzate—5.0 g, hydrolyzed animal tissues—5.0 g, glucose—40 g and agar—15.0 g. They were all sterilized using a steam-pressure autoclave at 121 °C, under a pressure of 1 atmosphere. Preparations containing humic substances and pure humic acids were added to the culture medium prepared in this way. Then, they were transferred to sterile plastic Petri dishes, with a diameter of 90 mm.

2.3. Effect on Colony Growth

In the first part of the experiment, the selected fungal isolates were grown on the SDA medium at 21 ± 1 °C. A fragment of mycelium from 10-day-old cultures was sampled with a preparation needle, to be inoculated centrally in to the solid SDA medium. The plates with the inoculated isolates were placed in incubators, protected from light, at 22 ± 1 °C. Colony growth observations were carried out every 5 days until day 20, by measuring the colony diameter in mm. The experiment was performed in four repetitions. The control consisted of cultures grown on substrates without the addition of preparations and pure humic acids. The results are presented as the size of the colony diameter expressed in mm and in the case of the 20th day of the culture as a percentage in relation to the control.

2.4. Effect on Spore Germination

In the second part of the experiment, the effect of the preparations containing humic substances and pure humic acids on the germination of spores of the fungal isolates was investigated. Spores from 3-week-old colonies were transferred with a scalpel to an aqueous solution. Under a microscope at 400× magnification, the titer (concentration) of the spores in the solution was determined using a Fuchs–Rosenthal chamber. The spore solution was diluted to 1.0 × 107 conidia/mL, which made it easier to observe the germinating spores. The number of spores in the field of view did not exceed 20–30. Approximately 1 mL of spore solution was pipetted onto the Sabouraud medium. The slides with the medium and spores were placed in incubators, at the temperature ranges outlined above. Germination observations were carried out after 24 and 48 h. A drop of lactophenol was added to the medium with spores, and it was covered with a cover glass. Then, in the field of view, the number of germinating spores per 100 observed conidia was counted. For each combination, i.e., EPF isolate–preparation, three replications were performed.

2.5. Statistical Analyses

The obtained results were analyzed statistically using the Statistica 13.3 TIBCO Software Inc. program. A one-way analysis of variance (ANOVA) and post-hoc Tukey test were performed. The calculated means were combined into homogeneous groups at the significance level α = 0.05. The standard deviation was calculated (SD).

3. Results

The present research on the development of the Metarhizium anisopliae fungus showed that products containing humic substances added to a culture medium, according to the recommended field dose, stimulated colony growth in relation to the control on the 10th, 15th and 20th day of observation (Table 2). On each date, statistically significant differences between the size of the colonies grown on the culture media with the products added and the control colonies were found. In the initial stage (day 5), only AmiAGRA stimulated colony growth, and the results were statistically significant. HumiAGRA and AlgoHUM limited the growth only slightly. However, on the 20th day of observation, the colonies of the M. anisopliae fungus were the largest when grown on the culture medium with the addition of AlgoHUM, but no statistically significant differences were found between the effects of the products. On the 5th day of observation, pure humic acids limited both the growth of the control colonies and the colonies grown on the culture medium with the addition of preparations containing humic substances. In the present research, the smallest size of the strain colony was recorded when grown on the culture medium with the addition of pure humic acids extracted from lignite. During each observation, a limiting effect of humic acids extracted from peat and added to the culture medium was found on the growth of the M. anisopliae fungus. Compared to the control, the larger sizes were observed on the culture medium with pure humic acids extracted from spent mushroom substrate, but this difference was not statistically significant.
The results of the present study indicated that when products containing humic substances were added to the culture medium, they stimulated the growth of Metarhizium flavoviride fungal colonies in relation to the control in each of the observation periods (Table 3). These differences were statistically significant. Growth was stimulated in the strongest way by HumiAGRA, with colonies reaching 67.7 mm on the 20th day of observation. In the present research, on day 5 of observation, it was found that pure humic acids added to the culture medium slightly stimulated the growth of M. flavoviride colonies compared to the control, but without any statistically significant effect. At each observation period, M. flavoviride colonies grown on the culture medium with pure humic acids extracted from peat were larger than the control colonies and, also, larger than those grown on the culture media with the addition of lignite or spent mushroom substrate.
The strongest limitation of the colony growth in relation to the control was on the culture medium with the addition of pure humic acids extracted from lignite, with statistically significant differences on the 15th and 20th days of observation.
On the 20th day of observation, it was found that products containing humic substances stimulated the growth of M. anisopliae fungal colonies more than that of M. flavoviride ones, and this difference was statistically significant. Pure humic acids limited the growth of the isolates, and M. flavoviride turned out to be a more sensitive species (Figure 1).
The proportion of EPF germinated spores after 48 h on the culture medium was higher than after 24 h (Table 4). In a statistically significant way, pure humic acids reduced spore germination more than products containing humic substances (Table 4, Figure 2). AlgoHUM after 24 h and HumiAGRA after 48 h reduced spore germination of the strains to the smallest extent. Pure humic acids extracted from peat after 24 h and from lignite after 48 h, on the culture medium, significantly lowered the number of germinated spores of the M. anisopliae fungus compared to the control. The germination of M. flavoviride spores after 24 h was most strongly inhibited by lignite humic acids added to the culture medium.

4. Discussion

The development of sustainable agricultural production involves the use of new plant protection products, with low toxicity to non-target organisms and high biodegradability. Resulting in a reduction in environmental damage and human health risks, agrotechnical and biological control methods are currently the safest alternative to chemical plant protection products [11,25,26,27]. In addition to reducing the number of pests, EPFs can also penetrate the tissue of plants, positively affecting their growth and development [2,16]. Some studies indicate that mycoinsecticides are more effective if they are used with the addition of appropriate excipients [2,28]. So far, humic substances have been studied mainly to determine their effects on bacterial and plant growth [29,30,31], while their effects on fungi and, in particular, on entomopathogenic fungi, have not been investigated enough [2,32,33].
Fedoseeva et al. [34] reported that humic substances act directly on living cells, with a wide range of biological effects, stimulating or inhibiting fungal growth. Tomaszewski et al. [35] and de Melo et al. [36] found that the addition of humic substances to a culture medium promoted an increase in fungal stability and resistance to stress. Raypuria et al. [37] reported that adjuvants added to preparations containing the M. anisopliae complex may increase the stability of its effectiveness. Testing the effect of three biofertilizers containing humic substances on the growth of the fungus M. anisopliae, Majchrowska-Safaryan and Tkaczuk [2] found that on the 20th day of observation all the biofertilizers limited the vegetative growth. However, there were no statistically significant differences in the sensitivity of the fungus M. anisopliae to the biofertilizers. Studying the effect of organic acids on the growth and development of EPFs, Li Holdom [38] concluded that they limit the vegetative growth of M. anisopliae mycelium. Hirose et al. [39] investigated the in vitro effect of three biofertilizers (EM-4, Multibion and Supermagro) on the M. anisopliae fungus and recorded a significant reduction in the vegetative growth and spore germination. The application of Multibion, one of the biofertilizers, resulted in the maximum negative effect on the vegetative growth of the fungus (43.59%), but no negative effects were found when the Supermagro biofertilizer was added to the culture medium.
Majchrowska-Safaryan and Tkaczuk [2] reported that the Rosahumus biofertilizer, added to a culture medium at the recommended field dose, on days 10 and 15 of observation did not limit the growth of the M. flavoviride fungus, but even stimulated it. Felizatti et al. [40] investigated the effect of various biopolymers, including humic acids, on the growth and germination of B. bassiana spores, and found no inhibitory effects. According to Fedosova et al. [34], the addition of humic substances to a culture medium stimulated A. alternata conidia production, with parallel inhibition of colony growth. When introduced into soil in the form of a biofertilizer, humic substances stimulate the release of organic acids from plant roots, and they become nutrients for beneficial microorganisms. This, in turn, enhances the growth of plant roots and their colonization by microorganisms, which is beneficial for plant and soil health [4,5,41]. Felizatti et al. [40] indicate that the inhibitory effect of humic acids is probably caused by a decrease in the pH value of the growth medium. Hirose et al. [39] found that Multibion and EM-4 significantly reduced the germination of M. anisopliae spores. Similarly, Gonazalez et al. [42] found that neem oil reduced the germination of M. anisopliae spores by 17.3%. In vitro studies conducted by Kaiser et al. [43] indicated the high potential of humic substances (above 90%) to protect B. bassiana spores against UV radiation, a physical factor limiting EPF conidia germination [44,45]. In addition, a significant increase in B. bassiana spore survival compared to the control was observed in field conditions 7 and 14 days after products containing humic acids were used [43].
The effect of humic substances and pure humic acids on the growth and germination of EPF spores has not been reported in the literature to any great extent. However, they are both increasingly being used as fertilizers, as they improve the condition of arable soils. Soils with high humic substances support plant growth, making them more tolerant to stress, healthier and able to produce higher yields with superior nutritional quality of the harvested food and feed [46]. In turn, EPFs inhabiting the soil environment form very complex relationships with other microorganisms and with plants, including crops [47,48,49].
In our own research, the commercial preparations used that contained humic substances had a positive effect on the growth of colonies and the germination of spores of the tested species of EPF. These preparations were characterized by higher pH values and also contained macroelements (N, K) and microelements, compared to pure humic acids [50]. The pH of pure humic acids was in the range of 4.5–5.3, which could have contributed to the acidification of the culture medium. Sellamuthu and Govindaswamy [51], when examining the effect of different doses of humic acids with potassium on the microbiology of the soil where sugar cane was grown, found that these treatments did not affect the growth of the bacterial population, but the total amount of fungi and actinomycetes increased. This was found to the greatest extent in the root rhizosphere, by as much as 150%.
The sustainable use of plant protection products is an increasingly important issue in crop production, especially in the context of restrictions on the amount of chemicals that can be used against pests. The present research indicates that environmentally compatible products that do not harm the natural environment, such as biofertilizers containing humic substances, could be applied simultaneously with EPFs, increasing their potential effectiveness in biological pest control [2,4,52,53]. Currently, microbiological preparations containing, among others, EPFs, constitute only 2% of the entire market of plant protection products in Poland [54]. However, it is expected that their range will expand due to the legal regulations introduced by the EU in 2022 regarding registration procedures for biopesticides containing microorganisms [55,56] and the opportunity for farmers to receive subsidies under the “Biological Pest Control” program introduced from 2023 [4,14].

5. Conclusions

Research on the impact of the combined use of humic substances with isolates of EPFs for the control of insects or mites is of great practical importance because it allows the use of selected strains that can be used together with commercially available biofertilizers in plant cultivation and protection toward sustainable agriculture. The conducted research showed that on the 20th day of culture (on average), preparations containing humic substances had a stimulating effect, while pure acids slightly limited the growth of the colonies of the tested isolates. The growth of M. anisopliae fungal colonies was most strongly stimulated by AlgoHUM, and M. flavoviride by HumiAGRA. The share of germinated spores after 48 h of contact with the substrate was higher than after 24 h, and more spores germinated on substrates with the addition of preparations containing humic substances than on pure humic acids.

Author Contributions

Conceptualization, A.M.-S. and C.T.; writing—original draft preparation A.M.-S., C.T. and M.W.; writing—review and editing, A.M.-S. and C.T. All authors have read and agreed to the published version of the manuscript.

Funding

The research carried out under research theme No. 159/23/B was financed by a science grant from the Ministry of Science and Higher Education.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Toader, G.; Chiurciu, V.; Mrierean, N.; Sevciuc, P.; Filip, V.; Burnichi, F.; Trifan, D.; Luxita, R.; CatalinIonut, E.; Toader, V.; et al. Economic advantages of using bacterial biopreparations in agricultural crops. In Proceedings of the Agrarian Economy and Rural Development—Realities and Perspectives for Romania, Bucharest, Romania, 11–13 October 2012; Volume 11, pp. 230–237. [Google Scholar]
  2. Majchrowska-Safaryan, A.; Tkaczuk, C. The effect of biofertilizers containing humic substances on the growth of Beauveria and Metarhizum fungi in vitro. Prog. Plant Prot. 2023, 63, 73–79. [Google Scholar] [CrossRef]
  3. Available online: http://rosahumus.pl/wyniki-badan/2021 (accessed on 10 February 2020).
  4. Holka, M.; Kowalska, J. The potential of adjuvants used with microbiological control of insect pests with emphasis on organic farming. Agriculture 2023, 13, 1659. [Google Scholar] [CrossRef]
  5. Da Silva, M.S.R.A.; dos Santos, B.M.S.; da Silva, C.S.R.A.; da Silva, C.S.R.A.; Antunes, L.F.S.; Santos, R.M.; Santos, C.H.B.; Rigobelo, E.C. Humic substances in combination with plant growth-promoting bacteria as an alternative for sustainable agriculture. Front. Microbiol. 2021, 12, 719653. [Google Scholar] [CrossRef] [PubMed]
  6. Gałązka, A.; Kocoń, A. Wpływ preparatów z mikroorganizmami pożytecznymi na liczebność i biomasę mikroorganizmów glebowych. Stud. I Rap. IUNG-PIB 2015, 45, 127–142. [Google Scholar]
  7. Pikuła, D. Rola substancji humusowych oraz innowacyjne produkty zwiększające ich zawartość w glebie. Stud. I Rap. IUNG-PIB 2016, 48, 81–93. [Google Scholar] [CrossRef]
  8. Lian, X.; Liao, S.; Yang, Y.; Zhang, X.; Wang, Y. Effect of pH or metal ions on the oil/water interfacial behavior of humic acid based surfactant. Langmuir 2020, 36, 10838–10845. [Google Scholar] [CrossRef]
  9. Kuźmiar, A.; Włodarczyk, K.; Gromadzka, P.; Siara, A.; Wolińska, A. Aktualny Stan Wiedzy na Temat Biopreparatów Stosowanych w Rolnictwie; Wydawnictwo KUL: Lublin, Poland, 2021; p. 32. [Google Scholar]
  10. Pylak, M.; Oszust, K.; Frąc, M. Review report on the role of bioproducts, biopreparations, biostimulants and microbial inoculants in organic production of fruit. Rev. Environ. Sci. Biotechnol. 2019, 18, 597–616. [Google Scholar] [CrossRef]
  11. Tkaczuk, C. Occurrence and Infective Potential of Entomopathigenic Fungi in Soils of Agrocenoses and Seminatural Habitats in the Agricultural Landscape; Scientific Dissertation No. 94; Wydawnictwo Akademii Podlaskiej: Siedlce, Poland, 2008. [Google Scholar]
  12. Vega, F.E.; Meyling, N.V.; Luangsa-ard, J.J.; Blackwell, M. Fungal entomopathogens. In Insect Pathology; Vega, F., Kaya, H.K., Eds.; Academic Press: San Diego, CA, USA, 2012; pp. 171–220. [Google Scholar]
  13. Quesada-Moraga, E.; González-Mas, N.; Yousef, M.; Garrido-Jurado, I.; Fernandez-Bravo, M. Key role of environmental competence in successful use of entomopathogenic fungi in microbial pest control. J. Pest Sci. 2023, 97, 1–15. [Google Scholar] [CrossRef]
  14. Runge, T.; Latacz-Lohmann, U.; Schaller, L.; Todorova, K.; Daugbjerg, C.; Termansen, M.; Liira, J.; Le Gloux, F.; Dupraz, P.; Leppanen, J. Implementation of eco-schemes in fifteen European Union Member States. EuroChoices 2022, 21, 19–27. [Google Scholar] [CrossRef]
  15. Sosnowska, D. Parasitic and antagonistic fungi in biological plant protection in Poland. Prog. Plant Prot. 2019, 59, 223–231. [Google Scholar] [CrossRef]
  16. Wasilewska-Nascimento, B. The underestimated potential of entomopathogenic fungi in potato cultivation. Ziemniak Polski 2021, 4, 40–48. [Google Scholar]
  17. Samson, R.A. Paecilomyces and some allied Hyphomycetes. Stud. Mycol. 1974, 6, 119. [Google Scholar]
  18. Humber, R.A. Manual of Techniques in Invertebrate Pathology. In Identification of Entomopathogenic Fung; Lacey, L.A., Ed.; Academic Press: London, UK, 2012; pp. 151–187. [Google Scholar]
  19. Schoch, C.L.; Seifert, K.A.; Huhndorf, S.; Robert, V.; Spouge, J.L.; Levesque, C.A.; Chen, W.; Fungal Barcoding Consortium. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Natl. Acad. Sci. USA 2012, 109, 6241–6246. [Google Scholar] [CrossRef]
  20. Kovač, M.; Gorczak, M.; Wrzosek, M.; Tkaczuk, C.; Pernek, M. Identification of entomopathogenic fungi as naturally occurring enemies of the invasive oak lace bug, Corythucha arcuata (Say) (Hemiptera: Tingidae). Insects 2020, 11, 679. [Google Scholar] [CrossRef] [PubMed]
  21. Altschul, S.F.; Madden, T.L.; Schäffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new 432 generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389–3402. [Google Scholar] [CrossRef] [PubMed]
  22. Becher, M.; Pakuła, K.; Czapliński, K. Soil Organic Matter Quality in Soils with Different Levels of Manure Fertilisation. Environ. Nat. Resour. J. 2020, 31, 17–23. [Google Scholar] [CrossRef]
  23. Becher, M.; Banach-Szott, M.; Godlewska, A. Organic matter properties of spent button mushroom substrate in the context of soil organic matter reproduction. Agronomy 2021, 11, 204. [Google Scholar] [CrossRef]
  24. Becher, M.; Tołoczko, W.; Godlewska, A.; Pakuła, K.; Żukowski, E. Fractional Composition of Organic Matter and Properties of Humic Acids in the Soils of Drained Bogs of the Siedlce Heights in Eastern Poland. J. Ecol. Eng. 2022, 23, 208–222. [Google Scholar] [CrossRef]
  25. Geiger, F.; Bengtsson, J.; Berendse, F.; Weisser, W.; Emmerson, M.; Morales, M.; Ceryngier, P.; Liira, J.; Tscharntke, T.; Winqvist, C. Persistent negative effects of pesticides on biodiversity and biological control potential on European farmland. Basic Appl. Ecol. 2010, 11, 97–105. [Google Scholar] [CrossRef]
  26. Fenibo, E.O.; Grace, N.I.; Matambo, T. Biopesticides in sustainable agriculture: A critical sustainable development driver governed by green chemistry principles. Front. Sustain. Food Syst. 2021, 5, 619058. [Google Scholar] [CrossRef]
  27. Ayilara, M.S.; Adeleke, B.S.; Akinola, S.A.; Fayose, C.A.; Adeyemi, U.T.; Gbadegesin, L.A.; Omole, R.K.; Johnson, R.M.; Uthman, Q.O.; Babalola, O.O. Biopesticides as a promising alternative to synthetic pesticides: A case for microbial pesticides, phytopesticides, and nanobiopesticides. Front. Microbiol. 2023, 14, 1040901. [Google Scholar] [CrossRef]
  28. Iglesias, L.; Groves, R.; Bradford, B.; Harding, R.; Nault, B. Evaluating combinations of bioinsecticides and adjuvants for managing Thrips tabaci (Thysanoptera: Thripidae) in onion production systems. Crop Prot. 2020, 142, 105527. [Google Scholar] [CrossRef]
  29. Zandonadi, D.B.; Canellas, L.P.; Rocha Façanha, A. Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H+ pumps activation. Planta 2007, 225, 1583–1595. [Google Scholar] [CrossRef]
  30. Xie, Y.; Gu, Z.; Herath, H.M.S.K.; Gu, M.; He, C.; Wang, F.; Jiang, X.; Zhang, J.; Zhang, Y. Evaluation of bacterial biodegradation and accumulation of phenanthrene in the presence of humic acid. Chemosphere 2017, 184, 482–488. [Google Scholar] [CrossRef] [PubMed]
  31. Kulikova, N.A.; Filippova, O.I.; Perminov, I.V. Protective activity of humic substances in wheat seedlings in water deficit conditions. Mosc. Univ. Soil Sci. Bull. 2018, 73, 76–80. [Google Scholar] [CrossRef]
  32. Fedoseeva, E.V.; Stepanov, A.; Yakimenko, O.; Patsaeva, S.; Freidkin, M.; Khundzhu, D.A.; Terekhova, V. Biodegradation of humic substances by microscopic filamentous fungi: Chromatographic and spectroscopic proxies. J. Soils Sediments 2019, 19, 2676–2687. [Google Scholar] [CrossRef]
  33. Fedoseeva, E.V.; Patsaeva, S.V.; Khundzhua, D.A.; Pukalchik, M.A.; Terekhova, V. Effect of exogenic humic substances on various growth endpoints of Alternaria alternata and Trichoderma harzianum in the experimental conditions. Waste Biomass Valorization 2020, 12, 211–222. [Google Scholar] [CrossRef]
  34. Fedoseeva, E.V.; Tereshina, V.M.; Danilova, E.A.; Ianutsevich, E.A.; Yakimenko, O.S.; Terekhova, V.A. Effect of humic acid on the composition of osmolytes and lipids in amelanin-containing phytopathogenic fungus Alternaria alternate. Environ. Res. 2021, 193, 110395. [Google Scholar] [CrossRef]
  35. Tomaszewski, J.E.; Schwarzenbach, R.P.; Sandar, M. Protein encapsulation by humic substances. Environ. Sci. Technol. 2011, 45, 6003–6010. [Google Scholar] [CrossRef]
  36. de Melo, B.A.G.; Motta, F.L.; Santana, M.H.A. Humic acids: Structural properties and multiple functionalities for novel technological developments. Mat. Sci. Engineer. 2016, 62, 967–974. [Google Scholar] [CrossRef]
  37. Raypuriya, N.; Das, S.B.; Bhowmick, A.K.; Vibha. Compatibility of Metarhizium anisopliae (Metchnikoff) Sorokin, with various adjuvants. J. Entomol. Zool. Stud. 2019, 7, 544–547. [Google Scholar]
  38. Li, D.P.; Holdom, D.G. Effects of nutrients on colony formation, growth, and sporulation of Metarhizium anisopliae (Deuteromycotina, Hyphomycetes). J. Invertebr. Pathol. 1995, 65, 253–260. [Google Scholar] [CrossRef]
  39. Hirose, E.; Neves, P.M.O.J.; Zequi, J.A.C.; Martins, L.H.; Peralta, C.H.; Moino, A. Effect of biofertilizers and neem oil on the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. Braz. Braz. Arch. Biol. Technol. 2001, 44, 419–423. [Google Scholar] [CrossRef]
  40. Felizatti, A.P.; Manzano, R.M.; Rodrigues, I.M.W.; das Graças Fernan, M.F.; Fernandes, J.B.; Forim, M.R. Encapsulation of B. bassiana in biopolymers. Improving microbiology of insect pest control. Front. Microbiol. 2021, 12, 704812. [Google Scholar] [CrossRef] [PubMed]
  41. Ren, H.; Islam, M.S.; Wang, H.; Guo, H.; Wang, Z.; Qi, X.; Zhang, S.; Guo, J.; Wang, Q.; Li, B. Effect of humic acid on soil physical and chemical properties, microbial community structure, and metabolites of decline diseased bayberry. Int. J. Mol. Sci. 2022, 23, 14707. [Google Scholar] [CrossRef] [PubMed]
  42. Gonzalez, D.M.E.; Valbuena, P.B.F.; Rivera, M.A.; Bustillo, P.A.E.; Chaves, B. Viabilidad del hongo Metarhizium anisopliae en mezcla con agroquimicos. Rev. Colomb. Entomol. 1996, 22, 31–36. [Google Scholar] [CrossRef]
  43. Kaiser, D.; Bacher, S.; Mène-Saffrané, L.; Grabenweger, G. Efficiency of natural substances to protect Beauveria bassiana conidia from UV radiation. Pest Manag. Sci. 2019, 75, 556–563. [Google Scholar] [CrossRef] [PubMed]
  44. Fargues, J.; Goettel, M.S.; Smits, N.; Ouedraogo, A.; Vidal, C.; Lacey, L.A. Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathogenic Hyphomycetes. Mycopathologia 1996, 135, 171–181. [Google Scholar] [CrossRef] [PubMed]
  45. Fernandes, É.K.K.; Rangel, D.E.N.; Moraes, Á.M.L.; Bittencourt, V.R.E.P.; Roberts, D.W. Variability in tolerance to UV-B radiation among Beauveria spp. Isolates. J. Invertebr. Pathol. 2007, 96, 237–243. [Google Scholar] [CrossRef]
  46. Pettit, R.E. Organic matter, humus, humate, humic acid, fulvic acid and humin: Their importance in soil fertility and plant health. CTI Res. 2004, 10, 1–15. [Google Scholar]
  47. Jaber, L.R.; Enkerli, J. Fungal entomopathogens as endophytes: Can they promote plant growth? Biocontrol. Sci. Technol. 2017, 27, 28–41. [Google Scholar] [CrossRef]
  48. Mantzoukas, S.; Grammatikopoulos, G. The effect of three entomopathogenic endophytes of the sweet sorghum on the growth and feeding performance of its pest, Sesamia nonagrioides larvae, and their efficacy under field conditions. Crop Prot. 2020, 127, 104952. [Google Scholar] [CrossRef]
  49. Canfora, L.; Tartanus, M.; Manfredini, A.; Tkaczuk, C.; Majchrowska-Safaryan, A.; Malusà, E. The impact of Beauveria species bioinocula on the soil microbial community structure in organic strawberry plantations. Front. Microbiol. 2023, 13, 1073386. [Google Scholar] [CrossRef]
  50. Schoebitz, M.; Lópes, M.; Serri, H.; Martinez, O.; Zagal, E. Combined application microbial consortium and humic substances to improve the growth performance of blueberry seedlings. J. Soil Sci. Plant Nutr. 2016, 16, 1010–1013. [Google Scholar] [CrossRef]
  51. Sellamuthu, K.M.; Govindaswamy, M. Effect of fertilizer and humic acid on rhizosphere microorganism and soil enzymes at an early stage of sugarcane growth. Sugar Tech. 2003, 5, 273–277. [Google Scholar] [CrossRef]
  52. Moino, A., Jr.; Alves, S.B. Efeito de imidacloprid e fipronil sobre Beauveria bassiana (Bals.) Vuill. e Metarhizium anisopliae (Metsch.) Sorok. e no comportamento de limpeza de Heterotermes tenuis (Hagen). An. Soc. Entomol. Brasil. 1998, 27, 611–619. [Google Scholar] [CrossRef]
  53. Quintela, E.D.; McCoy, C.W. Synergistic effect of imidacloprid and two entomopathogenic fungi on the behavior and survival of larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae) in soil. J. Econ. Entomol. 1998, 91, 110–122. [Google Scholar] [CrossRef]
  54. Ministry of Agriculture and Rural Development. Register of Plant Protection Products. Available online: https://www.gov.pl/web/rolnictwo/rejestr-rodkow-ochrony-roslin (accessed on 22 June 2023).
  55. Helepciuc, F.E.; Todor, A. Improving the authorization of microbial biological control products (MBCP) in the European Union within the EU Green Deal framework. Agronomy 2022, 12, 1218. [Google Scholar] [CrossRef]
  56. European Commission (EC). Micro-Organisms. Available online: https://ec.europa.eu/food/plants/pesticides/micro-organisms_en (accessed on 22 June 2023).
Sustainability 16 03616 g001
Figure 1. Average colony size on the 20th day of cultivation on media with the addition of preparations containing humic substances and pure humic acids. Notes: a,b—significant at α = 0.05, Tukey’s HSD.
Figure 1. Average colony size on the 20th day of cultivation on media with the addition of preparations containing humic substances and pure humic acids. Notes: a,b—significant at α = 0.05, Tukey’s HSD.
Sustainability 16 03616 g001
Sustainability 16 03616 g002
Figure 2. Germination of spores on media with the addition of preparations containing humic substances and pure humic acids. Notes: a,b—significant at α = 0.05, Tukey’s HSD.
Figure 2. Germination of spores on media with the addition of preparations containing humic substances and pure humic acids. Notes: a,b—significant at α = 0.05, Tukey’s HSD.
Sustainability 16 03616 g002
Table 1. Characteristics of preparations containing humic substances and pure humic acids used in the experiment.
Table 1. Characteristics of preparations containing humic substances and pure humic acids used in the experiment.
PreparationChemical CompositionFarm Crop Dose
AmiAGRA
Agraplant		pH—7.0–9.0, humic substances—60%, amino acids—70%, azot (N)—6%soil application: 0.5–1 kg ha−1 in 250–300 L of water; foliar application: 100 g ha−1 in 250–300 L of water
HumiAGRA
Agraplant		pH—8.0–10.0 humic substances—60%, potassium oxide (K2O)—8% soil application: 0.5–1 kg ha−1 in 250–300 L of water; foliar application: 100 g ha−1 in 250–300 L of water
AlgoHUM
Agraplant		pH—7.8–9.8, humic substances—28% including 50% humic acids, sea algae—50%100 g ha−1 in 250–300 L of water
PeatHumic acids extracted from low peat—Liwiec river valley, pH—4.5–5.01.2 g on dm3 of water
LigniteHumic acids extracted from lignite—the Bełchatów mine, pH—4.5–5.01.2 g on dm3 of water
SMSHumic acids extracted from spent mushroom substrate—large-area mushroom farms, Siedlce district, pH—4.5–5.31.2 g on dm3 of water
Table 2. Colony diameter of the fungus Metarhizium anisopliae during culture on media with the addition of preparations containing humic substances and pure humic acids.
Table 2. Colony diameter of the fungus Metarhizium anisopliae during culture on media with the addition of preparations containing humic substances and pure humic acids.
PreparationDiameter of Fungus Colony (mm)
5th Day10th Day15th Day20th Day
Preparations containing humic substances
AmiAGRA18.5 ± 1.00 a43.2 ± 0.95 a52.5 ± 1.73 a69.7 ± 0.50 a
HumiAGRA15.0 ± 2.94 b40.0 ± 0.96 b54.3 ± 0.47 a69.3 ± 0.47 a
AlgoHUM14.4 ± 1.91 cd37.3 ± 0.94 b54.0 ± 0.83 a70.3 ± 1.25 a
Pure humic acids
Peat14.5 ± 0.57 cd30.0 ± 0.82 c45.2 ± 1.25 d59.7 ± 4.91 c
Lignite13.2 ± 1.25 d32.3 ± 0.47 c47.6 ± 0.47 c62.6 ± 1.88 bc
SMS *13.7 ± 0.95 cd31.0 ± 0.82 c49.6 ± 0.47 b67.0 ± 1.87 b
Control15.3 ± 0.51 b31.2 ± 1.47 c46.4 ± 2.53 cd63.7 ± 1.93 bc
p-value0.000.000.000.00
F-value19,903.5614,337.38103,343.70282,408.00
* SMS—spent mushroom substrate; abcd—means within columns with the same lowercase letters are not significant at α = 0.05, Tukey’s HSD; ± standard deviation (SD).
Table 3. Colony diameters of the fungus Metarhizium flavoviride during culture on media with the addition of preparations containing humic substances and pure humic acids.
Table 3. Colony diameters of the fungus Metarhizium flavoviride during culture on media with the addition of preparations containing humic substances and pure humic acids.
PreparationDiameter of Fungus Colony (mm)
5th Day10th Day15th Day20th Day
Preparations containing humic substances
AmiAGRA17.2 ± 0.95 ab34.5 ± 0.58 b48.2 ± 2.21 c61.5 ± 1.73 c
HumiAGRA17.5 ± 1.29 a36.5 ± 2.38 a52.7 ± 2.98 a67.7 ± 3.30 a
AlgoHUM16.2 ± 1.50 bc33.5 ± 1.73 b50.0 ± 1.41 b64.3 ± 3.29 b
Pure humic acids
Peat15.5 ± 0.50 cd35.2 ± 0.95 ab51.2 ± 1.50 b62.5 ± 1.00 c
Lignite15.2 ± 1.00 cd29.0 ± 1.15 d42.0 ± 0.81 e53.6 ± 1.25 e
SMS *15.2 ± 0.50 cd31.7 ± 2.36 c44.2 ± 3.77 d55.0 ± 4.32 e
Control15.0 ± 0.63 d29.5 ± 1.37 d44.4 ± 3.38 d58.0 ± 1.90 d
p-value0.000.000.000.00
F-value31,142.9655,751.52160,742.40276,171.30
* SMS—spent mushroom substrate; abcde—means within columns with the same lowercase letters are not significant at α = 0.05, Tukey’s HSD; ± standard deviation (SD).
Table 4. Germination of fungal spores on media with the addition of preparations containing humic substances and pure humic acids (in % in relation to the control).
Table 4. Germination of fungal spores on media with the addition of preparations containing humic substances and pure humic acids (in % in relation to the control).
Species/
Preparation		Metarhizium anisopliaeMetarhizium flavoviride
after 24 hafter 48 hafter 24 hafter 48 h
Preparations containing humic substances
AmiAGRA90.5 b94.6 b90.2 c94.8 c
HumiAGRA91.3 b96.6 b91.8 b96.3 b
AlgoHUM92.2 b94.8 b92.3 b96.2 b
Pure humic acids
Peat76.3 c84.2 c70.4 e85.7 d
Lignite74.7 c86.2 c76.4 d83.5 d
SMS *77.1 c88.6 c78.3 d84.1 d
Control100 a100 a100 a100 a
p-value0.000.000.000.00
F-value1,059,559693,375.00913,424.60817,020.10
* SMS—spent mushroom substrate; abcde—means within columns with the same lowercase letters are not significant at α = 0.05, Tukey’s HSD.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Majchrowska-Safaryan, A.; Tkaczuk, C.; Wrzosek, M. The Effect of Humic Substances on the Colony Growth and Conidial Germination of Entomopathogenic Fungi from the Genus Metarhizium. Sustainability 2024, 16, 3616. https://0-doi-org.brum.beds.ac.uk/10.3390/su16093616

AMA Style

Majchrowska-Safaryan A, Tkaczuk C, Wrzosek M. The Effect of Humic Substances on the Colony Growth and Conidial Germination of Entomopathogenic Fungi from the Genus Metarhizium. Sustainability. 2024; 16(9):3616. https://0-doi-org.brum.beds.ac.uk/10.3390/su16093616

Chicago/Turabian Style

Majchrowska-Safaryan, Anna, Cezary Tkaczuk, and Marta Wrzosek. 2024. "The Effect of Humic Substances on the Colony Growth and Conidial Germination of Entomopathogenic Fungi from the Genus Metarhizium" Sustainability 16, no. 9: 3616. https://0-doi-org.brum.beds.ac.uk/10.3390/su16093616

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop