Orchid
The endophytes in orchids are mostly specific fungi of Basidiomycota, such as Sebacinaceae, Ceratobasidiaceae, and Tulasnellaceae (Zettler and Corey 2018; Li et al. 2021; Wang et al. 2021). In addition, some endophytes from the root, especially
During a screening of EHB in the root-associated endophytic fungi of
Meanwhile, the fungal DNA was extracted by the CTAB method (Stenglein and Balatti 2006). The PCR amplification of 16S rDNA region for the genomic DNA was performed using primers of ER10 (5’-GGCGGACGGGTGAGTAA-3’) and ER11 (5’-ACTGCTGCCTCCCGTAG-3’) to examine its harboring endohyphal bacteria. It was carried out in a T100TM thermal cycler (BIO-RAD, USA) with the following parameters: initial denaturing for 30 s at 94°C, then 30 cycles at 94°C for 10 s, annealing at 55°C for 45 s, then at 72°C for 30 s, with the final extension for 10 min at 72°C (Cheng et al. 2022). The PCR products were visualized and displayed on 1% agarose electrophoresis gel under ultraviolet illumination.
The purified EHB was inoculated on LB agar, cultured at 28°C for 24 h, and the colony morphology, color, and other traits were observed. Scanning electron microscopy (SEM) was used to observe the morphology and determine the size of EHB. The EHB was inoculated in LB medium, cultured at 28°C, 200 rpm, for 20 h, and the cultures were centrifuged at 8,000 rpm for 5 min to collect the cells. After fixing in 2.5% glutaraldehyde solution for 2–4 h, EHB’s cells were washed three times with 0.1 M phosphate buffer (pH 6.8), dehydrated with 30%, 50%, 70%, 90%, and 100% ethanol in turn, for 15–20 min each, replaced twice with isoamyl acetate, and processed in the vacuum freeze dryer for 3 h. After drying, the samples were mounted on stubs, sputter-coated with gold-palladium, and finally examined with SEM (VEGA 3 SBU, Tescan, China) (Jiang et al. 2019b).
Since the EHB appeared as
To determine the PGP ability for the EHB strain YZSR384, it was inoculated into the demineralized LB medium at 28°C, 200 rpm overnight, and adjusted to an appropriate concentration (OD600 = 1.0). The previously mentioned sterile rice seeds were soaked in the bacterial culture for one day, and the PGP ability was assayed. The demineralized LB medium was used as a control.
The PGP-related IAA production was determined by the Salkowski method, which resulted in the color changes of the EHB broth (Fig. 4A). The results indicated that EHB YZSR384 could produce IAA. In the quantitative analysis of IAA production, it appeared that IAA was produced in the highest concentration on the sixth day, reaching 23.361 μg/ml after inoculation. Then the concentration began to decline (Fig. 4B).
Genome properties of EHB
Feature | |
---|---|
Genome size (bp) | 4,215,636 |
GC content (%) | 43.51 |
Total number of genes | 4444 |
Number of CDSs | 4216 |
tRNA genes | 87 |
rRNA genes | 30 |
tmRNA genes | 1 |
Genomic islands | 4 |
Genes allocated to Uniprot | 4,157 |
Genes allocated to Refseq | 4,180 |
Genes allocated to KEGG | 2,330 |
Genes allocated to Pfam | 3,761 |
Genes allocated to GO | 3,359 |
Genes allocated to COG | 2,816 |
Genes associated with plant growth-promoting (PGP) traits for EHB
PGP activity | Gene | Function | Chromosome location |
---|---|---|---|
IAA production | Tryptophanyl-tRNA synthetase | 1218699–1217707 |
|
Tryptophan synthase subunit alpha | 2371911–2371108 |
||
Tryptophan synthase subunit beta | 2373106–2371904 |
||
Phosphoribosylanthranilate isomerase | 2373734–2373087 |
||
Indole-3-glycerol phosphate synthase | 2374491–2373739 |
||
Anthranilate phosphoribosyltransferase | 2375500–2374484 |
||
Anthranilate synthase | 2377019–2375472 |
||
Phosphate solubilization | Alkaline phosphatase | 283609–285360 |
|
Alkaline phosphatase | 621083–619695 |
||
Alkaline phosphatase | 1018051–1016675 |
||
Phosphate starvation-inducible protein | 2615049–2614090 |
||
Alkaline phosphatase synthesis transcriptional regulatory protein | 2978117–2977395 |
||
Two-component sensor histidine kinase | 3236753–3238354 |
||
Phosphate ABC transporter ATP-binding protein | 2577585–2576803 |
||
Phosphate ABC transporter permease | 2579310–2578426 |
||
Phosphate ABC transporter permease | 2580239–2579310 |
||
Phosphate ABC transporter substrate-binding protein | 2581210–2580308 |
||
Siderophore production | Isochorismatase | 552212–552754 |
|
Isochorismate synthase | 3153313–3151898 |
||
2,3-dihydroxybenzoate-AMP ligase | 3289855–3288236 |
||
2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase | 3291080–3289884 |
Endohyphal bacteria (EHBs) exist in a variety of fungi which species and number are also variable within the same genus or family (Hoffman and Arnold 2010; Shaffer et al. 2016). EHBs in
The siderophores synthesized by
EHB can assist the host fungi in surviving in unfavorable environments (Ghignone et al. 2012; Salvioli et al. 2016) and perform specific biological functions living outside the fungus (Ruiz-Herrera et al. 2015; Pakvaz et al. 2016; Aslani et al. 2018). It indicates that EHB is also a potential biological rich resource, which is worthy of further exploration and development. The present EHB could be used for the development of PGP candidates.