1. Introduction

Nutritional composition and antioxidant activity due to the abundance of phenolic compounds in leaves extracts are beneficial to human health [14,15]. The growing area of V. myrtillus is native to Europe, but this species is also found in temperate and sub-Arctic regions around the world. Bilberries are widely abundant and easily found in the forests of Lithuania, Latvia, Finland, and Norway. Well-drained, moist, acidic soils are best for this species, but it can also grow in very acidic soils (pH 4.5–6). Thus, the adaptation and secondary metabolism of V. myrtillus plants can be synergistically affected by microbial endophytes, the benefits and potential of which are, in many cases, unknown in wild forest plants. In addition, some endophytes have shown a good ability to colonize host plant tissues; therefore, bacteria have a beneficial effect on plant growth by providing plants with the necessary nutrients or bioactive compounds [16,17,18,19]. Many beneficial microorganisms from different plant species and environments have recently been identified that can act as sources of new bioactive compounds and can therefore be used in the medical, agricultural or food industries. Numerous microbiological and ecological studies have shown that plant endophytes and their products may be promising candidates as a biological control measure.

2. Biodiversity of Endophytic Bacteria in Bilberry Leaves

A total of 25 genetically distinct endophytic bacteria were isolated from the leaves of V. myrtillus according to the sequence data of the 16S rRNA gene (Table 1). BLAST and phylogenetic analysis of 16S rRNA gene sequences revealed that the endophytic bacterial isolates were 99.58–100% similar to the sequences available in the NCBI GenBank. The 16S rDNA nucleotide sequences were submitted to GenBank and assigned accession numbers MZ469297 to MZ469321. The genetic identity among the sequences was found to be 100% in 18 bacteria strains. The 16S rRNA gene sequences demonstrated that strains Bil-LT1_1, Bil-LT1_2, Bil-LT4_7, Bil-LV3_1, and Bil-FIN2_3 were identical to several Bacillus spp. species from NCBI; however, due to genetic similarity within the Bacillus spp. species, precise identification of the isolates should be performed for further studies. The same situation was observed for strains Bil-LT4_8 and Bil-NOR3_14, which were related to Micrococcus sp., and strains Bil-Bil-2_5, Bil-Bil-2_6, and Bil-NOR3_11, which were related to Staphylococcus sp. One strain, Bil-FIN2_7, was related to Lysinibacillus spp. Three strains (Bil-LV3_4, LV3_6, and NOR3_18) with minor nucleotide polymorphisms were closely related to Paenibacillus spp. Strains of endophytic bacteria, such as Rothia amarae (Bil-LT4_1), Paenibacillus tundrae (Bil-LV3_3), Kocuria kristinae (Bil-FIN2_9, Bil-FIN2_13), Weissella hellenica (Bil-FIN2_10), Micrococcus terreus (Bil-FIN2_12), Corynebacterium freneyi (Bil-NOR3_13), Pseudomonas monteilii (Bil-NOR3_15), Sporosarcina aquimarina (Bil-NOR3_16), and Paenibacillus xylanexedens (Bil-NOR3_17), were identified in bilberry leaves.

Table 1. Comparative matches for the closest phylogenetic genotypes (according NCBI records) obtained for the culturable bacteria isolates based on profile of 16S rRNA gene.

Isolate	Accession Number in NCBI	Identity Accessions, According NCBI	Sequence Length, bp (Identity, %)
Bil-LT1_1	MZ469297	Bacillus halotolerans MK517597.1 B. mojavensis MF040286.1 B. velezensis MT634548.1 B. axarquiensis GU568194.1 B. subtilis AB526464.1	1437 (100)
Bil-LT1_2	MZ469298	Bacillus simplex LK391525.1 Peribacillus butanolivorans CP050509.1	1431 (100)
Bil-LT4_1	MZ469299	Rothia amarae MG905369.1	1400 (99.79)
Bil-LT4_3	MZ469300	Bacterium strain MTL8-4 MH151301.1	1439 (99.58)
Bil-LT4_7	MZ469301	Bacillus zhangzhouensis MN826587.1 B. pumilus CP054310.1 B. safensis KJ542766.1 B. stratosphericus KY203662.1	1420 (100)
Bil-LT4_8	MZ469302	Micrococcus sp. MG132043.1 M. luteus AJ409096.1	1398 (100)
Bil-LV3_1	MZ469303	Bacillus sp. strain MK736127.1 B. aryabhattai MN515130.1 B. megaterium MF988696.1	1427 (100)
Bil-LV3_3	MZ469304	Paenibacillus tundrae HF545335.1	1431 (100)
Bil-LV3_4	MZ469305	Paenibacillus sp. MK290403.1	1435 (99.65)
Bil-LV3_6	MZ469306	Paenibacillus sp. MG758020.1	1450 (99.86)
Bil-FIN2_3	MZ469307	Bacillus cereus MN068934.1 B. thuringiensis CP050183.1	1439 (100)
Bil-FIN-2_5	MZ469308	Staphylococcus warneri CP038242.1 S. pasteuri MW433878.1	1437 (100)
Bil-FIN2_6	MZ469309	Staphylococcus warneri CP038242.1 S. pasteuri MW433878.1	1437 (100)
Bil-FIN2_7	MZ469310	Lysinibacillus macrolides MH542661.1 L. xylanilyticus KP644237.1 L. fusiformis FJ641020.1	1427 (100)
Bil-FIN2_9	MZ469311	Kocuria kristinae KX055834.1	1384 (100)
Bil-FIN2_10	MZ469312	Weissella hellenica CP042399.1	1447 (100)
Bil-FIN2_12	MZ469313	Micrococcus terreus KJ781899.1	1385 (100)
Bil-FIN2_13	MZ469314	Kocuria kristinae KX055834.1	1384 (100)
Bil-NOR3_11	MZ469315	Staphylococcus sp. KM253075.1	1431 (100)
Bil-NOR3_13	MZ469316	Corynebacterium freneyi EF462412.1	1393 (99.86)
Bil-NOR3_14	MZ469317	Micrococcus sp. KX350143.1 M. luteus MN826463.1	1385 (100)
Bil-NOR3_15	MZ469318	Pseudomonas monteilii CP013997.1	1422 (100)
Bil-NOR3_16	MZ469319	Sporosarcina aquimarina MK726086.1	1433 (100)
Bil-NOR3_17	MZ469320	Paenibacillus xylanexedens CP018620.1	1436 (99.79)
Bil-NOR3_18	MZ469321	Paenibacillus sp. KR055031.1	1427 (99.86)

The biodiversity of endophytic bacteria in bilberry leaves from the countries of the Baltic-Nordic region (Lithuania, Latvia, Finland, and Norway) was investigated and is shown in Figure 1. Endophytic bacteria isolated from bilberry leaves belonged to the phyla Firmicutes 64%, Actinobacteria 32%, and Proteobacteria 4%. Bacteria of the families Bacillaceae, Paenibacillaceae, and Micrococcaceae were the most frequently isolated and identified in blueberry leaves. Staphylococcaceae, Lactobacillaceae, Pseudomonaceae, Corynebacteriaceae, and Planococcaceae strains were identified in one or more cases.

Figure 1. Biodiversity of endophytic bacteria in bilberry leaves of the Baltic-Nordic region’s forests—Lithuania (LT), Latvia (LV), Finland (FIN) and Norway (NOR).

Endophytic bacteria isolated from Norwegian bilberry leaves belonged to Firmicutes 1%, Actinobacteria 12%, and Proteobacteria 4% phyla. Meanwhile, bacteria in Finnish and Lithuanian leaf samples belonged to Firmicutes (20% and 16%, respectively) and Actinobacteria (12% and 8%, respectively). Only Firmicutes phylum bacteria were isolated from the Latvian samples. Bacteria of the Bacillaceae family were isolated and identified in the bilberry leaves of Lithuania, Latvia, and Finland, but were not found in plant samples from Norway. Bacteria belonging to six different families isolated from leaves samples were collected from the northern countries—Finland and Norway (Figure 2).

This entry is adapted from the peer-reviewed paper 10.3390/f12121647