The analysis of variance demonstrated that PGPRs inoculant, Cold stratification periods and their interaction effects on germination traits were significant (Table 3, P < 0.05). Seed inoculation significantly increased seed germination, germination speed, mean germinayion time, seed vigor index, and root and shoot length of Caucasian whortleberry.
Effect of bacterial inoculation and cold stratification on germination percentage (GP)
The percentage of seed germination was 9, 7. And 6.5 I the seeds which inoculated by P. putida, E. cloacea and B. subtilis, 9, 7 and 6.5%, respectivelyThe non inoculated seeds did not germinate (Fig. 1). P. putida increased the seed germination percentage more than B. subtilis and E. cloacea.
Many researches have shown that PGPR inoculations can increase the germination traits in many seeds of woody species such as Acacia senegal (Singh et al. 2011). Crataegus pseudoheterophylla (Fatemeh et al. 2014), Abies religiosa and A. hickelii (Zulueta-Rodríguez et al. 2015), Corylus avellana (Rostamikia et al. 2016), Acasia mearnsii (São José et al. 2019), and Abies nordmanniana (Garcia-Lemos et al. 2020).
The effect of different bacteria is various on seed germination and plant growth (Basu et al. 2021). In this context, it can be stated pseudomonas strains by affecting different parts of the seed, playing a role in the biosynthesis and secretion of pklant growth hormones and reducing the ratio of abscisic to gibberellin, which breaks embryo dormancy and stimulates germination (Miransari and Smith 2014). It is a well-known that inoculation of seeds with PGPB strains increases the uptake of water and nutrient and effective synthesis of auxins, gibberellins, and reduces inhibitors such as ABA in the seed endocarp (de Andrade et al. 2023).
The results showed the mixed inoculion of bacteria had better effect compared to individual inoculation on germination traits. The application of cmixed inoculum (B. subtilis, E. colacea and P. putida) increased the germination percentage to 14 showing the synergic effect of bacteria. Similar to our findings, the co-inoculation seeds of C. pseudoheterophylla with Azotobacter chroococcum, Azospirillum lipoferum, Pseudomonas fluorescens and Bacillus subtilis increased seed germination (Fatemeh et al. 2014).
The germination was 6.5, 16, 18, 40 and 40.5% in seeds, which stratified for 1, 2, 3, 4, and 5 months, respectively while non-stratified seeds did not germinate in the control (Figure.1). a period of 1, 2 and 3 months of cold stratification was insufficient to induce percentage of germination in V. arctostaphyos seeds. While 4 and 5- month cold stratification resulted significant increase in seed germination percentage, which indicates the presence of physiological dormancy in the seed, which controls a significant part of the seed dormancy in this species. the highest germination (70%) of V. membranaceum seeds was obtained by 28 days stratification at 1–3°C (Barney, Shafii, and Price 2001) and the maximum germination of seeds of V. myrtillus and V. vitis-idaea were 62 and 100%, respectively, in light at 20:10°C and 25:15°C (Baskin et al. 2000). The chilling reduces the content of abscisic acid (ABA) by increasing the content of gibberellic acid. These changes are made simultaneously as creating a balance between two hormones (Bewley and Black 2013). It is possible that the cold factor, in addition to stimulating the endogenous gibberellin synthesis, activates other stimuli that increase the percentage of seed germination (Chen et al. 2009). Also, during Cold stratification, a significant increase in the level of phosphate pathway enzymes coincided with breaking seed dormancy.
The results of our study indicated the highest germination percentage was observed in seeds co-inoculated by three bacteria followed by 5 -months cold stratification period with 58.5% that did not differ significantly (p > 0.05) from the GP (57.5%) of the coi-noculation of three bacteria followed by 4-month CS period pretreatment (Fig. 1).
Efect of bacterial inoculation and cold stratification on germination speed (GS) and mean germination time (MGT)
In general, germination speed is considered one of the most important factors affecting plant establishment (Chen et al. 2007). The results showed that the inoculation of PGPRs, period of CS and the interaction of these factors had a significant effect on the germination speed (Table 1). when the seeds were inoculated with PGPR individually and mixed, the germination rate increased. So that, the highest germination speed (2.26 n/d) was observed in seeds co-inoculated by three bacteria followed by 5- months cold stratification (Fig. 2). Earlier studies have indicated that bacterial inoculums are able to increase the germination speed. In this context, the highest germination speed (4.82 n/d) was obtained in Crataegus pseudoheterophylla seeds using the combination of all bacterial of Azotobacter chroococcum, A. lipoferum, Pseudomonas fluorescens and Bacillus subtilis (Fatemeh et al. 2014). these findings may be due to the increased synthesis of hormones like gibberellins and better synthesis of auxins, which have a positive effect on germination speed (Ahemad and Kibret 2014).
The results of our study showed, the minimum of mean germination time (MGT) was observed in seeds co-inoculated followed by 5-month CS period with 13.84 day. The mean germination time (MGT) was 25.50, 28.26 and 32.92 days when the seeds were inoculated by P. putida, E. colacea and B. subtilis, respectively. Co-inoculation decreased the mean germination time (MGT) compared in individual inoculation (Fig. 3). P. putida and B. subtilis are among the most important phosphate solubilizes (Kalayu 2019).
In CS treatments (without inoculation), the increase in CS period (from 1 to 5 months) led to a gradual decrease in MGT. These results confirmed the importance of the cold stratification in accelerating the growth of the embryo, increasing the germination speed and reducing the germination period.
Effect of bacterial inoculation and cold stratification on root and shoot length and seed vigor index
Different strains of rhizobacteria had variable effects on root and shoot length in various ecotype assays. Individual inuculation of bacteria, the maximum root and shoot length were observed in P. putida with 8.74 and 12.30 mm, respectively (Fig. 4). Similary, Rostamikia et al. (2016) reported the increase in root and shoot of hazelnut (Corylus avellana L.) inoculated with Pseudomonas putida. Chaín et al. (2020) also observed significant root and stem growth in seedlings of Eucalyptus grandis inoculated with two Pseudomonas strains after 50 days. The most important characteristics of Pseudomonas spp. is production of auxines, siderophores, hydrogen cyanide (HCN), ammonia (NH3), exo-polysaccharides, and also solubilization of phosphate (Costa-Gutierrez et al. 2022). Plant hormones such as auxins, gibberellins, and cytokinins produced by rhizobacteria play an important role in cell division, and root and shoot elongation (Chaín et al. 2020).
In each bacterial treatment, the increase in CS period led to a gradual elongation in root and shoot length. tThe highest root length was allocated to the seeds co-inoculated by bacteria followed by 5-month cold stratification with 17.98 mm (Fig. 4). The same trend was observed in shoot length. The maximum shoot length was observed when the seeds co-inoculated by bacteria followed by 5 months CS with 23.25 mm (Fig. 5).
Seed vigor is the most important indicator for determining seed quality, the potential for fast and uniform emergence of seedlings (Wen et al. 2018). In this study, seed germination index was significantly affected by individual and combined inoculation of different bacterial strains. The results of variation bacterial treatments of V. arctotaphylos are presented in Fig. 6. the highest value of seed germination index was obtained in the treatment of seeds inoculated with the combination of three bacteria. Among inoculated bacteria in state on individual applications, P. putida had with 18.30 the highest effect on SVI. Seed vigor index was improved with increasing CS periods in both PGPRs inoculation and un-inoculation. So that, the highest value of seed germination index with 24.12 belonged to the appliction of combined inoculation three bacteria (B. subtilis, E. colacea and P. putida) with 5 months CS treatment (Fig. 6). The reason for the increase in the seed vigor index can be related to the movement of nutrient reserves, the activity and resynthesis of some enzymes, the start of the resynthesis of RNA and DNA, and the rapid growth of the embryo after the elimination of the barriers to germination