Fungal Endophytes of Vitis vinifera—Plant Growth Promotion Factors

Endophytes are microorganisms that live asymptomatically inside plant tissues. They are beneficial to their host in many aspects, especially as a defense against foreign phytopathogens through the production of a variety of metabolites. These substances can serve as sources of new natural products for medicinal, agricultural, and industrial purposes. This article is focused on endophytic fungi from Vitis vinifera. The purpose of the research was their isolation and identification during the Vitis vinifera growing season. Subsequently, the isolates were tested for the production of biotechnologically interesting metabolites (siderophores, antioxidants, and antifungal compounds). In total, 24 endophytic fungi were isolated, the most represented genus was Cladosporium sp. The results of the test for antioxidant and antifungal properties, as well as siderophore production, have shown that the population of Vitis vinifera endophytic microscopic fungi could serve as a promising source of metabolites with a wide range of applications.


Introduction
The grapevine (Vitis vinifera) is one of the most economically important crops, being used mainly for wine production (approximately 80% of harvested wine grapes is used for this purpose). Grapes and other parts of Vitis vinifera contain a number of health promoting metabolites [1]. As with other plants, the tissues of the grapevine are inhabited by various types of microorganisms. These organisms can be epiphytic, i.e., superficial, or colonizing internal tissues, i.e., endophytic [2]. The interactions between endophytes and their plant hosts are diverse. Plants provide protection and endophytic microorganisms are capable of producing useful metabolites that increase nutrient uptake, induce resistance to pathogens, increase tolerance to osmotic stress, heavy metals, xenobiotic contaminants, and other forms of abiotic stress [3]. Most endophytes are represented by bacteria, but microscopic fungi and yeasts also form a significant part of the endophytic population. Endophytes are isolated from a variety of plant species, and almost all studied plant species have been found to host at least one endophytic microorganism [4]. Colonization of the host plant with up to a hundred different species is no exception. Geographic location, season, climate, and type of plant tissue are among the factors that affect species composition and frequency of endophyte colonization [5,6].
Endophytes act as important sources of structurally unique bioactive natural metabolites with a wide biotechnological potential. They represent an attractive source of natural products that can be used in agriculture, industry, and medicine [7][8][9][10][11][12][13]. The formation of

Samples
Samples of Muller Thurgau, Pinot Gris, Pinot Noir and Riesling Rheinhessen grapevine varieties were collected from two different vineyards within the Czech Republic, Kutna Hora (49.9336 N, 15.2889 E; grapevine grown according to the principles of organic farming) and Prague (50.0690 N, 14.4454 E; conventionally grown grapevine). Three different experimental plants located at different sites within the vineyards were selected for continuous sampling during the entire vegetation year. The sampling of leaves and canes as lignified stems of the plants was carried out in January, May, August and October 2019 in approximate amounts between 3 and 10 g of leaves, depending on the sampling season (leaves were not sampled in January due to their fall during the autumn), and canes were collected in approximate amounts of 50 g. Berries were sampled in September 2019 in an amount of 500 g, only from the Prague vineyard (strong storms in Kutna Hora region ruined the crops and sampling was not possible). The samples were stored at −80 • C before processing in the laboratory. Characterization of the samples from which were fungal endophytes is provided in Table 1.

Fungal Endophytes Isolation and Cultivation
The plant material was surface sterilized by sequential immersion in 0.625% aqueous sodium hypochlorite with a drop of Tween 80 (7 min), followed by 70% aqueous ethanol (3 min). After these procedures, the samples were rinsed four times with sterilized water (15 min). The surface-sterilized tissues were homogenized and used to inoculate YGC medium (yeast extract glucose chloramphenicol agar) and incubated at 20 • C for 72 h or more.

Molecular Genetic Identification of Endophytes
Genomic DNA was isolated from pure fungus culture by using the ArchivePure DNA Yeast and Gram-+ Kit (5 PRIME, Hamburg, Germany). Subsequently, the nuclear ribosomal ITS1-5, 8S-ITS2 region was determined for all strains according to Kolařík et al. [26]. Due to the low resolution of the ITS region in some fungal species, the sequencing of other sections was made to clarify the identification. Elongation factor 1 alpha (EF1α) was amplified and sequenced using primers EF-728F/EF-986R and EF1-983F/EF1-2218R according to Kolařík et al. [26]. The partial β-tubulin (TUB2) gene was amplified using T1/T2 according to Píchová et al. [27]. The sequences obtained were manually cut from unreadable sections and the highest probability of the acquired sequence was searched in the GenBank database.

Determination of Siderophores Production of the Isolates
The method of Marques et al. [28] was followed to determine siderophore production. Fungal cultures were inoculated on Chrome azurol S (CAS) agar, and cultivated at 28 • C for 7 days. After cultivation, the color change (blue to yellow) was evaluated and scaled (0 = blue medium surface, no siderophore production; 1 = 30% yellow medium surface-low siderophore production; 2 = 60% yellow medium surface-medium siderophore production, 3 = yellow medium surface, high siderophore production (Figure 1.)).
al. [27]. The sequences obtained were manually cut from unreadable sections and the highest probability of the acquired sequence was searched in the GenBank database.

Determination of Siderophores Production of the Isolates
The method of Marques et al. [28] was followed to determine siderophore production. Fungal cultures were inoculated on Chrome azurol S (CAS) agar, and cultivated at 28 °C for 7 days. After cultivation, the color change (blue to yellow) was evaluated and scaled (0 = blue medium surface, no siderophore production; 1 = 30% yellow medium surface-low siderophore production; 2 = 60% yellow medium surface-medium siderophore production, 3 = yellow medium surface, high siderophore production (Figure 1.)).

Figure 1.
Fungal endophyte cultured on CAS agar with high siderophore production activity (color change from blue to yellow).

Determination of Antioxidant Activity of the Isolates
The fungal endophyte isolates were grown in PDB medium at 30 °C for 7 days with constant shaking. The antioxidant activity of the supernatant of the filtered culture was determined according to Fidler and Kolářová [29]. The analyses were performed on the microtiter plates in three parallels for each sample. An aliquot of 100 μL of the sample was pipetted together with 200 μL of DPPH at a concentration of 52 mg L −1 (in methanol) in the wells. Distilled water was used as a blank. The plate was incubated in the dark for 15 min. The absorbance was measured at 517 nm. The results of the analysis were expressed as the percent decrease in the discoloration of the solution against the blank. The results were expressed as an ascorbic acid (AA) equivalent, which was chosen as an analytical standard in the concentration range of 2.5-25 mg L −1 .

Determination of Antifungal Activity of the Isolates
Antifungal activity was tested on PDA agar according to Bell et al. [30]. Two wells (7.5 mm diameter) were excavated in the agar at the same distance from the center. One of the wells was filled with agar with a grown fungal endophyte and the other with agar containing a fungal phytopathogen. These plates were cultivated at 28 °C for 7 days. Testing was carried out with three fungal phytopathogens-Botrytis cinerea DBM 1246, Fusarium solani CCF 2967, and Mucor plumbeus CCF 2626. The phytopathogenic culture itself served as a control sample. Antifungal activity was displayed by slowing or stopping the growth of a fungal phytopathogen in the vicinity of the growth of an endophytic fungus. The degree of fungal antagonism was evaluated on a scale of 5-1 (5-the endophyte completely outgrows the phytopathogen; 4-the endophyte colonizes 2/3 of the medium surface; 3-the endophyte and the phytopathogen both colonize half of the medium surface ( Figure 2); 2-phytopathogen Figure 1. Fungal endophyte cultured on CAS agar with high siderophore production activity (color change from blue to yellow).

Determination of Antioxidant Activity of the Isolates
The fungal endophyte isolates were grown in PDB medium at 30 • C for 7 days with constant shaking. The antioxidant activity of the supernatant of the filtered culture was determined according to Fidler and Kolářová [29]. The analyses were performed on the microtiter plates in three parallels for each sample. An aliquot of 100 µL of the sample was pipetted together with 200 µL of DPPH at a concentration of 52 mg L −1 (in methanol) in the wells. Distilled water was used as a blank. The plate was incubated in the dark for 15 min. The absorbance was measured at 517 nm. The results of the analysis were expressed as the percent decrease in the discoloration of the solution against the blank. The results were expressed as an ascorbic acid (AA) equivalent, which was chosen as an analytical standard in the concentration range of 2.5-25 mg L −1 .

Determination of Antifungal Activity of the Isolates
Antifungal activity was tested on PDA agar according to Bell et al. [30]. Two wells (7.5 mm diameter) were excavated in the agar at the same distance from the center. One of the wells was filled with agar with a grown fungal endophyte and the other with agar containing a fungal phytopathogen. These plates were cultivated at 28 • C for 7 days. Testing was carried out with three fungal phytopathogens-Botrytis cinerea DBM 1246, Fusarium solani CCF 2967, and Mucor plumbeus CCF 2626. The phytopathogenic culture itself served as a control sample. Antifungal activity was displayed by slowing or stopping the growth of a fungal phytopathogen in the vicinity of the growth of an endophytic fungus. The degree of fungal antagonism was evaluated on a scale of 5-1 (5-the endophyte completely outgrows the phytopathogen; 4-the endophyte colonizes 2/3 of the medium surface; 3-the endophyte and the phytopathogen both colonize half of the medium surface ( Figure 2); 2-phytopathogen colonizes 2/3 of the medium surface; 1-the phytopathogen completely outgrows the endophyte).

Statistical Analysis
Dixon's Q test was performed to detect outliers in datasets obtained by the determination of antioxidant activity (the determination was performed in five parallels. The deviation of the five determinations was less than 5%). The determination of ability to produce siderophores and antifungal activity was performed in three parallels.

Statistical Analysis
Dixon's Q test was performed to detect outliers in datasets obtained by the determination of antioxidant activity (the determination was performed in five parallels. The deviation of the five determinations was less than 5%). The determination of ability to produce siderophores and antifungal activity was performed in three parallels.

Fungal Endophytes Characterization and Molecular Genetic Identification
For canes and leaves, a total of 24 endophytic microscopic fungi belonging to 14 fungal genera were isolated from both vineyards. Six isolates were obtained during the winter from both conventional and organic farming localities, ten isolates were obtained from the spring collection, with the majority of endophytes originating from organically grown plants, four fungal endophytic species were isolated from summer samples from the organic farming region, and four isolates came from the autumn sampling, from both farming system localities. The genus Cladosporium was represented by two species, Cladosporium cladosporioides and Cladosporium herbarum. The further more represented genera were Didymella sp., Aspergillus sp., Aureobasidium sp. and Alternaria sp. All five isolates obtained from the berries belong to Penicillium sp., specifically to the species Penicillium cructosum (for details, see Table  2).

Fungal Endophytes Characterization and Molecular Genetic Identification
For canes and leaves, a total of 24 endophytic microscopic fungi belonging to 14 fungal genera were isolated from both vineyards. Six isolates were obtained during the winter from both conventional and organic farming localities, ten isolates were obtained from the spring collection, with the majority of endophytes originating from organically grown plants, four fungal endophytic species were isolated from summer samples from the organic farming region, and four isolates came from the autumn sampling, from both farming system localities. The genus Cladosporium was represented by two species, Cladosporium cladosporioides and Cladosporium herbarum. The further more represented genera were Didymella sp., Aspergillus sp., Aureobasidium sp. and Alternaria sp. All five isolates obtained from the berries belong to Penicillium sp., specifically to the species Penicillium cructosum (for details, see Table 2).

Production of Siderophores
Siderophore production was established for 83% of isolates from the winter biomass collection, in 60% of isolates from the spring sampling, in 75% of isolates from summer, in 50% of fungal endophytes species being isolated from autumn leaves and cane samples, and in all endophytes isolated from berries. The highest ability to produce siderophores (degree '3') was detected for endophytes from winter sampling and organically grown canes, in particular for Diatrype stigma (Z-MT-KH-S1 isolate) and Aspergillus niger (Z-RM-KH-S2 isolate), and for endophytes originating from berries, i.e., Penicillium crustosum (MT-M4 isolate, RR-M2 isolate, RS-M2 isolate).

Antioxidant Activity
The ability to produce antioxidants into the medium was identified in all endophytes isolated from winter canes, in 80% of endophytes isolated from the spring biomass collection, in 75% of isolates originating from the summer and autumn V. vinifera biomass, and in all of the isolates from berries. The content of antioxidants expressed as ascorbic acid (AA) equivalent was determined in the range of 4.8-21.8 mg AA L −1 for the winter isolates, 0-17.6 mg AA L −1 for the spring isolates, 0-9.3 mg AA L −1 for the summer isolates, 0-7.5 mg AA L −1 for the autumn endophytes from leaves and canes and 9.3-23.9 mg AA L −1 for the autumn isolates from berries. The endophytes with the highest antioxidant production were isolates from berries, i.e., Penicillium crustosum (RR-M2 isolate and RS-M2 isolate), with 23.9 and 19.1 mg AA L −1 , respectively, and Aspergillus pseudodeflectus (Z-RM-KH-S1 isolate) from winter organically farmed canes (21.8 mg AA L −1 ). For details, see Table 2.

Antifungal Activity
The highest degree of antagonism, level '5' explaining the highest antagonism where the endophyte completely outgrows the phytopathogen, was against F. solani and was observed for Penicillium crustosum (MT-4, RR-M2, RS-M2) isolated from berries. All these three endophytic isolates also showed significant antifungal activity against B. cinerea (level '4').
The other relatively strong antagonist of B. cinerea DBM 4111 was the endophyte Lophiostoma corticola (L-RS-KH-L4) isolated from organically grown leaves collected in summer. For the phytopathogen F. solani CCF 2967, the other highly effective endophytes were Aspergillus niger (Z-RM-KH-S2) isolated from organically farmed winter canes and Aspergillus fumigatus (L-MT-KH-L5) isolated from organically farmed summer leaves. For Mucor plumbeus CCF 2626, the highest antagonist was Aspergillus niger (Z-RM-KH-S2) isolated from organically grown winter canes. For details, see Table 2.

Discussion
Recently, endophytic microorganisms and their products have been attracting the attention of the scientific community as a relatively poorly understood source of a wide range of chemically diverse natural substances potentially usable in biotechnology, pharmaceutical and food industry. The increased attention for studying endophytic populations is based on the desire to produce non-chemical based solutions. Comparing endophytic populations in-situ in terms of the presence of individual microorganisms or the formation of their metabolites is challenging due to the many factors (altitude, temperature, total precipitation, rhizosphere composition, or pesticide use) that affect these populations. Despite this, the methods for testing the endophytic isolates in laboratory conditions are well established.

Fungal Endophytes Characterization and Molecular Genetic Identification
Another degree of variability in the endophyte composition is the physiological state of the host plant itself, its growth phase, and the tissue from which the sample is taken. Thus, even isolates from the same geographical location may be diametrically different [31][32][33][34]. This variability was confirmed in this work, where isolates of fungal endophytes from two vineyards of different farming systems were examined. The Kutna Hora vineyards grow grapevines according to the principles of organic farming, and the Prague vineyards grow their grapevines in a conventional way. The total number of fungal endophytes isolated from canes and leaves from organically grown plants was approximately three times higher than the number of endophytes isolated from conventional vineyards, which is consistent with previous studies [35,36]. This difference could be related to the use of chemical or organic fertilizers and herbicides that directly affect microorganisms or alter the physiology of the host plant [37,38]. The response of endophytic microbial communities to these external products is very beneficial for comparing organic and conventional agriculture, and further research could go in this direction.
Precipitation is one of the main abiotic factors that affect the density of endophytes in the host plant [39][40][41]. The proportion of endophytes in the leaves of trees that have been protected from rain is lower than in the leaves of identical trees, but unprotected from rainfall. Suryanarayanan et al. dealt with this issue in the rainforest environment during the rainy season and drought. In all tested leaf samples of Bauhinia racemosa, Ixora nigricans, Erythroxylon monogynum and Elaeodendron glaucum, an increased representation of the endophytic community was detected during the rainy season [40]. The more abundant colonization of plants by fungal microorganisms at higher precipitation rates may be related to the consequent increased number of endophytes in the host plant. Precipitation is also one of the major types of endophytic spore transmission. So far, there is a little information on where endophyte spores are produced, where they can hibernate and what the mode of transmission is. R. parkeri sporulates prolifically on Contarinia midge galls on Douglas fir needles, and there were measured 1200 spores/mL in water dripping from a heavily galled branchlet. R. parkeri and its anamorph also sporulate in the fall in abscised needles. The spore masses of this endophyte are produced in mucilage, which is indicative of water transmission. Further, newly flushed needles in the spring do not become infected until they are rained on in the fall. From these findings we could suggest that the association Agriculture 2021, 11, 1250 8 of 13 between higher endophyte counts and moisture is not accidental [41]. If we follow the number of cane and leaf isolates obtained from individual periods of the Vitis vinifera growing year, the predominance of spring sampling is evident (Table 1.). May 2019 (spring sampling) was well above the long-term precipitation average. August 2019 (summer sampling) was only slightly below this average and October 2019 (autumn sampling) was quite average, which corresponds to a lower endophytic proportion (Figure 3). However, heavy rain and storms could completely destroy the grapevine crop, which unfortunately occurred in autumn 2019 in the Kutna Hora vineyards. These natural phenomena made the sampling of berries from this biodynamic vineyard in September 2019 impossible. The amplification of ITS rDNA and subsequent comparison of the obtained sequence with the database is currently the most widely used method for the identification of fungal endophytes [42,43]. All 29 isolates belonging to 15 genera were successfully identified, and all of them belonged to the Ascomycota phylum. This phylum significantly predominates in the proportion of fungal endophytes in Vitis vinifera, regardless of the geographical location of the host plant [34,44]. The most abundant genera were Penicillium sp., Cladosporium sp., Didymella sp., Aspergillus sp., Aureobasidium sp., and Alternaria sp. Alternaria sp. and Cladosporium sp. are one of the most abundant endophytes of Vitis vinifera [34,43], which is in accordance with our results.

Production of Siderophores
Siderophores have received great attention in medicine, biotechnology, and environmental research due to their high affinity and specificity for Fe 3+ . The only fungal endophyte isolates from canes and leaves with a high ability to produce siderophores came from the winter period of 2019. The ability to form these compounds has been declining since winter, with only low or zero production activity in summer and autumn. The highest result of siderophore production in winter can be explained by the reduced movement of nutrients in the soil due to low temperatures and therefore iron deficiency in both the endophyte and the host plant and the increased need for uptake of these nutrients by other mechanisms [45]. Diatrype stigma and Aspergillus niger were isolates with the highest detected siderophore production activity. In this paper, the production of siderophores by the genus Diatrype was proved for the first time. With regard to the high activity of production of these compounds identified, it would be interesting to pay further attention to the species of this microbial genus in the research. Aspergillus species are well-researched producers of siderophores, serving as a model organism to elucidate the biosynthesis, absorption, and degradation of these secondary metabolites [46]. Three berry isolates also showed a high ability to produce siderophores. All of these isolates belong to Penicillium crustosum, which is in agreement with the findings in the literature that this genus is capable of siderophore production [47]. In a study on the characterization of siderophores produced by endophytes from Cymbidium aloifolium, the genus Penicillium was found to be the best producer of these compounds [48].  The amplification of ITS rDNA and subsequent comparison of the obtained sequence with the database is currently the most widely used method for the identification of fungal endophytes [42,43]. All 29 isolates belonging to 15 genera were successfully identified, and all of them belonged to the Ascomycota phylum. This phylum significantly predominates in the proportion of fungal endophytes in Vitis vinifera, regardless of the geographical location of the host plant [34,44]. The most abundant genera were Penicillium sp., Cladosporium sp., Didymella sp., Aspergillus sp., Aureobasidium sp., and Alternaria sp. Alternaria sp. and Cladosporium sp. are one of the most abundant endophytes of Vitis vinifera [34,43], which is in accordance with our results.

Production of Siderophores
Siderophores have received great attention in medicine, biotechnology, and environmental research due to their high affinity and specificity for Fe 3+ . The only fungal endophyte isolates from canes and leaves with a high ability to produce siderophores came from the winter period of 2019. The ability to form these compounds has been declining since winter, with only low or zero production activity in summer and autumn. The highest result of siderophore production in winter can be explained by the reduced movement of nutrients in the soil due to low temperatures and therefore iron deficiency in both the endophyte and the host plant and the increased need for uptake of these nutrients by other mechanisms [45]. Diatrype stigma and Aspergillus niger were isolates with the highest detected siderophore production activity. In this paper, the production of siderophores by the genus Diatrype was proved for the first time. With regard to the high activity of production of these compounds identified, it would be interesting to pay further attention to the species of this microbial genus in the research. Aspergillus species are well-researched producers of siderophores, serving as a model organism to elucidate the biosynthesis, absorption, and degradation of these secondary metabolites [46]. Three berry isolates also showed a high ability to produce siderophores. All of these isolates belong to Penicillium crustosum, which is in agreement with the findings in the literature that this genus is capable of siderophore production [47]. In a study on the characterization of siderophores produced by endophytes from Cymbidium aloifolium, the genus Penicillium was found to be the best producer of these compounds [48].

Antioxidant Activity
There is growing evidence of oxidative damage to biomolecules by free radicals. These injuries could cause much tissue harm. Antioxidants are considered highly effective in defending tissue against damage caused by reactive oxygen species [49]. Fungal endophytes can be a potentially very good source of antioxidants [50] which has been confirmed in isolates in this study. The isolate with the highest antioxidant activity (21.7 mg AA L −1 ) of canes and leaves was Aspergillus pseudodeflectus. Arora and Chandra [51] investigated the antioxidant activity of the genus Aspergillus, specifically Aspergillus fumigatus. The data have shown that this microorganism can serve as a promising source of antioxidant compounds. Our results show an even higher antioxidant activity for the fungal endophyte Aspergillus pseudodeflectus than was mentioned in the article. Other studies also mention the high antioxidant activity of endophytes of Aspergillus sp. and the possibilities of further use of these properties [52,53]. Therefore, this fungal genus could serve to more easily adjust the production and purification of natural antioxidants.
In general, the proportion of genera with antioxidant activity in Vitis vinifera is relatively high, which correlates with the assumption of the formation of similar secondary metabolites between the host plant and its endophytes. Grapevine itself is an important source of antioxidants, especially phenolic substances.

Antifungal Activity
The fungal endophytes of Vitis vinifera could have an antagonistic effect on some important phytopathogens. Studies mapping this antifungal ability of endophytic communities are essential to shape pest control strategies but also to potential production of high-quality agricultural products. In the case of grapevine, one of its most common pathogens is the fungus Botrytis cinerea, which causes Botrytis bunch rot. The most effective antifungal agents against this phytopathogen are the endophytes Alternaria sp. and Epicoccum sp. Both are also promising biocontrol agents against Plasmopara viticola, another important source of Vitis vinifera diseases [34]. The antifungal ability found against Botrytis cinerea in Alternaria and Epicoccum isolates in this research work was expressed by the degree of antagonism at level '3', which could be expressed as 50%. The mentioned genera did not show above average activity even against the other two tested phytopathogens Fusarium solani and Mucor plumbeus. Fusarium solani is an important plant pathogen that most often causes rot in the root system. It has the ability to penetrate cell walls and therefore cause plant tissue to rot [62]. Mucor plumbeus is associated with the growth of fungi in cereals, rice, soybeans, nuts, fruits, herbs, and others [63].
The highest degree of antagonism (level '4') against Botrytis cinerea was detected in isolates from canes and leaves in only one endophyte, Lophiostoma corticola. This is the first paper to show the ability of this fungus to produce antifungal compounds. The ability of the Lophiostoma genus has been shown to produce metabolites that are effective only against pathogenic bacteria [64,65]. However, Lophiostoma corticola had a degree of antagonism of level '1' against Fusarium solani and Mucor plumbeus and therefore zero antifungal activity. For such a questionable result, it would be ideal to perform antifungal tests with other phytopathogens to detect the antifungal activity of this genus. When we continue with the antifungal results from isolates connected with canes and leaves, the highest activity against Fusarium solani was detected in two isolates of the genus Aspergillus, Aspergillus niger and Aspergillus fumigatus. Aspergillus niger was the only species to show the highest activity also against Mucor plumbeus. Antifungal activity against the phytopathogens Giberella zeae, Thanatephorus cucumeris and six other nonpathogenic microscopic fungi was detected in the endophyte Aspergillus fumigatus isolated from Hyoscyamus muticus [66]. Aspergillus flavus, an endophyte of Lannea coromandelica, showed high antifungal activity against Candida albicans and Malassezia pachydermis. Aspergillus niger isolated from the same host plant showed a moderate ability to form antifungal metabolites against the mentioned phytopathogens [67]. From the mentioned studies, it can be concluded that the antifungal activity of the genus Aspergillus is high, which is in accordance with our results. The genus Penicillium is known for its antifungal effect on Botrytis cinerea [7,68,69] and also shows this effect on Fusarium sp. [70,71]. Penicillium crustosum isolates from berries confirmed these findings, as they exhibited high antifungal activity against both Botrytis cinerea and Fusarium solani.

Conclusions
The population of endophytic fungi of Vitis vinifera has proven to be a promising source of growth-promoting and protective properties useful for the plant. Further studies are needed to investigate endophytic fungi in detail as a potential source of secondary metabolites. As it is a very poorly researched source of metabolites, it would be interesting to use the findings of this research work, choose the most productive endophytic species, and conduct detailed research. A closer focus on the formation of siderophores could be very useful in conjunction with enhancement of plant growth and biocontrol against phytopathogens. The study of antifungal metabolites could be used to develop effective biopesticides that could be a more environmentally friendly option for both the plant and the environment.