Various types of relationship between two or more organisms are well known among plants and in the animal kingdom. However, little is known about the relationship and interactions between fungi and bacteria. There are three most common types of these relationships. Bacteria can live in the same environment as fungi where both organisms live close to each other but not in direct contact. This kind of relationship is common in various environments like forests, fermented food or animal tissues affected by diseases (Frey-Klett et al. 2011). More complicated type of bacterial-fungal association is mixed biofilms, where bacteria may live on fungal hyphae and are held by the extracellular matrix formed from molecules secreted by both organisms (Donlan and Costerton 2002). This kind of coexistence can be found in medical equipment (Pierce 2005) as well as in some infections (Hogan et al. 2007) or mycorrhizal systems (Sarand et al. 1998). The third type of interactions between these two groups of organisms is endosymbiosis when bacteria live inside fungal cells and do not produce any specific structures. It is the most complex type of relationship and often, in contrast to free-living fungi-associated bacteria it is impossible to cultivate such bacteria outside a fungal host. The presence of unculturable endosymbiotic bacteria was described mostly in cells of arbuscular mycorrhizal fungi belonging to
The aim of this work was to identify bacteria associated with eight
The identification of the associated organisms was performed using whole DNA isolated from fungal strains. Fresh fungal mycelia were harvested from 25 mm2 square area of the solid medium of 7 days old fungal colonies, then suspended and rubbed in 1 ml of 0.1 M MgSO4 in sterile glass mortars. Suspension in a volume of 100 μl of each fungal strain was used for DNA isolation using PowerSoil DNA Isolation Kit (MO-BIO Laboraories, Inc., USA) according to the manufacturer’s protocol. DNA was purified as described above.
The identification of bacteria was performed using a molecular cloning technique. Products of 16S rRNA PCR were purified with PCR SureClean Plus (Bioline LTD., UK) kit. Cloning was carried out with Zero Blunt PCR Cloning Kit (Life Technologies, USA) according to manufacturer’s protocol. The ligation mixture was prepared in 50:1 insert:vector ratio (250 ng of purified product), the ligation step was 30 minutes long. Competent
Accessing numbers of
NCBI accessing number | Parent strain | Number of clones | The most similar strains in NCBI database | |
---|---|---|---|---|
Strain name | Accessing number and similarity (%) | |||
KR779716 | PxS; PBo6S | 2 | JF905617 (99) | |
KR779718 | PBo6S | 1 | CP009215 (99) | |
KR779719 | PBo6S | 1 | EF658675 (100) | |
KR779720 | PB8 | 1 | KX379256 (100) | |
KR779721 | PxS; PBo6S; PB7’96 | 4 | CP027427 (99) | |
MH424523 | P112 | 2 | KU060300 (100) | |
KR779722 | PBo6S; PB63S; P234; PxS | 5 | HQ264096 (100) | |
MH424529 | PB7’96 | 1 | LC371258 (100) | |
KR779723 | P234 | 1 | CP005586 (99) | |
KR779725 | PB7’96 | 1 | KT185191 (99) | |
KR779726 | P234; PB8; P112; PBo6S | 8 | MG995009 (100) | |
MH424530 | P112 | 2 | JX505089 (99) | |
MH424531 | PB8 | 1 | MH368091 (99) | |
KR779727 | PxS | 1 | EU071473 (100) | |
KR779728 | PBo6S; PB7’96 | 4 | NR_044185 (99) | |
KR779729 | PxS | 1 | JF274870 (99) | |
KR779730 | PxS | 1 | KC545358 (99) | |
MH424533 | P234 | 1 | KT805418 (99) | |
MH424534 | PB8 | 1 | KJ510213 (100) | |
MH424535 | P112 | 1 | MG214549 (99) | |
MH424536 | P234; PxS | 2 | KU921566 (99) | |
KR779731 | PxS; P112; PB7’96; P234; PB8 | 9 | NR_115049 (99) | |
KR779732 | P112; P234; PB8 | 5 | CP014132 (99) | |
MH424568 | PxS | 1 | KT184899 (100) | |
MH424569 | PxS | 1 | KT591476 (99) | |
MH424570 | P112 | 1 | LT899997 (100) | |
MH424571 | PB8 | 1 | KP183056 (99) | |
KR779733 | PB8; PBo6S | 2 | CP025986 (99) | |
MH426745 | PBo6S | 1 | KF755496 (100) | |
KR779734 | PUSAS; PBo6S; P234; PB7’96; P112 | 11 | CP026544 (100) | |
MH424572 | PBo6S | 1 | JX912460 (99) | |
KR779735 | PxS; P112 | 2 | NR_113367 (99) | |
MH424575 | P112 | 1 | FJ957593 (99) | |
KR779736 | PB7’96; P112; PxS; P234; PB63S; Pbo6S | 11 | KP183061 (99) | |
KR779737 | PxS; PB8; P234; P112 | 5 | CP025261 (100) | |
KR779738 | PB7’96 | 1 | KT695813 (100) | |
MH424593 | PB7’96 | 2 | MF838663 (100) | |
MH424580 | PB8; PB63S | 2 | KY939757 (100) | |
MH424594 | P112 | 1 | CP015639 (100) | |
MH424599 | PB7’96 | 1 | CP023272 (100) | |
KR779740 | P112; PB63S; PB7’96 | 4 | KP967499 (100) | |
MH427201 | PB63S; PBo6S | 3 | JN541150 (100) | |
KR779742 | PUSAS | 11 | CP029579 (100) | |
MH427368 | PUSAS | 21 | CP027440 (100) | |
KR779743 | PUSAS | 31 | CP027763 (100) | |
MH427381 | PUSAS | 1 | CP027766 (100) | |
KR779745 | PxS | 1 | KX139193 (99) | |
MH427585 | PxS | 1 | CP025839 (99) | |
MH427654 | PB8 | 1 | CP020459 (99) | |
KR779746 | PxS | 1 | AB856218 (98) | |
KR779747 | PxS | 1 | NR_113383 (99) | |
KR779748 | PxS | 1 | S | CP014616 (100) |
KR779749 | PBo6S | 1 | CP018838 (99) | |
KR779750 | PB8 | 1 | KP345929 (100) | |
KR779751 | PB8 | 1 | MH057260 (100) | |
KR779752 | P234 | 1 | MH014970 (100) | |
KR779753 | P234 | 1 | CP009769 (100) | |
KR779754 | P234 | 1 | AB649006 (100) | |
KR779755 | P234 | 1 | KF441681 (99) | |
KR779756 | P112 | 1 | KT716268 (100) | |
KR779757 | PB7’96 | 1 | KU495919 (100) | |
MH427999 | P234; PBo6S; PB8; PB63S; PxS; PB7’96; P112 | 35 | KP967473 (100) | |
MH428000 | PB63S, PB7’96; P112 | 3 | KX260804 (99) | |
MH428038 | P234 | 1 | HQ598999 (99) | |
MH428102 | PB7’96 | 1 | HQ598816 (99) | |
MH428220 | P112 | 1 | NR_109462 (99) | |
MH428377 | P112 | 1 | NR_148618 (99) | |
MH428379 | P234 | 1 | CP026616 (99) | |
MH428572 | P234 | 1 | KM070563 (99) | |
MH428659 | PBo6S | 1 | LC002811 (99) | |
MH428674 | PBo6S | 1 | KP262513 (99) | |
MH428833 | PB7’96 | 1 | MF113252 (99) |
A number of small bacteria-like organisms were observed inside and outside of cells of all analyzed oyster mushroom strains. They were visible as small green or yellow-green rods. The microscopic image is shown in Fig. 1. The bacteria-like organisms were marked with white arrows. Several of the cells observed were motile inside of fungal hyphae what is presented in materials published online (PxS –
Microscopic picture of analyzed oyster mushroom cells treated with fluorescent dyes under the fluorescent microscope. Strains: a) P234, b) PBo6S, c) PB8, d) P112, e) PB63S, f) PUSAS, g) PB7’96, h) PxS. Bacteria-like organisms, marked with white arrows, are visible as small green or yellow-green rods against fungal cells.
Bacteria-like organisms microcolonies growing deeply in TSAt medium after 10 days long cultivation. Strains: a) P234, b) PBo6S, c) PB8, d) P112, e) PB63S, f) PUSAS, g) PB7’96, h) PxS.
As a result of PCR performed with DNA obtained from precipitant and 799f and 1492r primers, a 700 bp product was obtained (Fig. 3). Product that size is characteristic only for bacterial DNA as it is a part of the bacterial 16S rRNA gene. For comparison, the reaction with maize DNA provided two bands: one of 1100 bp which is characteristic for plant mitochondrial DNA and second of size about 700 bp, characteristic for bacteria. The reason for this phenomenon is that maize cultivar used in experiment carry endophytic bacteria in its tissues (Pisarska and Pietr 2015).
Agarose gel electrophoresis showing products of PCR with 799f and 1942r primers of analyzed oyster mushroom, maize cv. Cyrkon (
The total number of 307 sequences was obtained as a result of the sequencing of plasmid clones. The detailed number of identified clones with the most similar strains from NCBI database is presented in Table II. The species or genus was determined for 233 of them and for the remaining 74 sequences we were not able to identify higher taxonomic level than domena of
Number and identification of clones obtained from analyzed
Genus | Similarity based on 700 bp sequence of the 16S rRNA gene | Number of clones | Origin | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
The most closely related strain in NCBI | NCBI accession number(% of similarity) | USA | Japan | Poland | |||||||
PUSAS | PB8 | PB7’96 | PBo6S | P234 | PxS | PB63S | P112 | ||||
CP029579 (100) | 64 | 11 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
CP027440 (100) | 21 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |||
CP027763 (100) | 31 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |||
CP027766 (100) | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |||
KP967473 (100) | 38 | 0 | 6 | 8 | 4 | 6 | 4 | 2 | 5 | ||
KX260804 (99) | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | |||
KP183061 (100) | 14 | 0 | 0 | 1 | 1 | 2 | 2 | 2 | 3 | ||
113367 (99) | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | |||
FJ957593 (99) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |||
MG995009 (100) | 13 | 0 | 2 | 0 | 1 | 2 | 0 | 0 | 3 | ||
JX505089 (99) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | |||
CP005586 (99) | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |||
KT185191 (99) | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |||
MH368091 (99) | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |||
CP026544 (100) | 12 | 2 | 2 | 1 | 3 | 2 | 0 | 0 | 2 | ||
JX912460 (99) | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |||
CP025261 (100) | 12 | 0 | 2 | 0 | 0 | 1 | 1 | 0 | 1 | ||
MF838663 (100) | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | |||
KY939757 (100) | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | |||
CP015639 (100) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |||
KT695813 (100) | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |||
CP023272 (100) | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |||
CP014132 (99) | 9 | 0 | 1 | 0 | 0 | 2 | 0 | 0 | 2 | ||
KT184899 (100) | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | |||
KT591476 (99) | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | |||
LT899997 (100) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |||
KP183056 (100) | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |||
NR_115049 (99) | 9 | 0 | 2 | 1 | 0 | 2 | 3 | 0 | 1 | ||
Acidovorax sp. clone M_KL_81_14 | KP967499 (100) | 4 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 2 | |
Acidovorax sp. clone CSC28 | JN541150 (100) | 3 | 0 | 0 | 0 | 1 | 0 | 0 | 2 | 0 | |
CP027427 (99) | 6 | 0 | 0 | 1 | 2 | 0 | 1 | 0 | 0 | ||
KU060300 (100) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | |||
KT805418 (100) | 6 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
KJ510213 (100) | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |||
KC545358 (99) | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | |||
MG214549 (99) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |||
KU921566 (99) | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | |||
HQ264096 (100) | 6 | 0 | 0 | 0 | 1 | 1 | 2 | 1 | 0 | ||
LC371258 (100) | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |||
NR_044185 (99) | 5 | 0 | 0 | 2 | 2 | 0 | 0 | 0 | 0 | ||
EU071473 (99) | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | |||
CP02598 (99) | 3 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | ||
KF755496 (100) | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |||
CP009215 (99) | 3 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | ||
EF658675 (100) | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |||
KX379256 (100) | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |||
CP020459 (99) | 3 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | ||
KX139193 (99) | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | |||
CP025839 (99) | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | |||
HQ598999 (99) | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
HQ598816 (99) | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |||
NR_109462 (99) | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | ||
NR_148618 (99) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |||
CP026616 (99) | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
KM070563 (99) | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |||
LC002811 (99) | 2 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | ||
KP262513 (99) | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |||
AB856218 (98) | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
NR_113383 (99) | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
CP014616 (100) | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
CPO18838 (99) | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | ||
KP345929 (100) | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | ||
MH057260 (100) | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | ||
MHO 14970 (100) | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
CP009769 (100) | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
AB649006 (100) | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
KF441681 (99) | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
KT716268 (100) | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | ||
KU495919 (100) | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | ||
JF274870 (99) | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
MF113252 (99) | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | ||
Number of diferent clones | X | X | X | 5 | 15 | 16 | 17 | 18 | 17 | 8 | 17 |
Number of genus | X | X | 32 | 2 | 9 | 13 | 13 | 15 | 13 | 5 | 12 |
Number of unidentified clones of | X | X | 74 | 0 | 6 | 10 | 14 | 10 | 15 | 7 | 12 |
Phylogenetic tree of identified bacteria associated with
The PCR reactions designed for detection of
Agarose gel electrophoresis showing products of PCR with Cu1AF and Cu2R primers and analyzed oyster mushroom DNA. Visible 140 bp band represents amplified LMCO genes. M – marker; B – blind control.
Yara et al. (1999, 2006) reported that bacteria or bacteria-like organisms could interact with only one strain of
The origin of identified