Screening and identification of epiphytic and soil bacteria beneficial in the control of postharvest apple disease

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A total of 2,027 bacterial isolates were obtained from the phyllosphere of apple, strawberry, raspberry, and grape plants, as well as from soil associated with apple, strawberry, and raspberry crops. Screening using a dual-plate confrontation assay identified 1,678 isolates with antagonistic activity against Botrytis cinerea , the causative agent of grey mold. Subsequent testing on apple fruit enabled the selection of 13 isolates exhibiting the highest biocontrol efficacy. These isolates were identified as belonging to the species Serratia inhibens (22ZK, 24PK, 132M), Pseudomonas laurylsulfativorans (131M), Pseudomonas alvandae (133M), Pantoea agglomerans (T16/8, M13/07), Pantoea allii (T14/15, T08/33, T08/48), and Pantoea ananatis (T5/1, T14/1A, T14/8). The five most effective isolates (T5/1, T14/15, T16/8, T14/1A, and M13/07) reduced disease severity by 62.4% to 78.6%. Further antagonism assays against revealed that the selected isolates, besides B. cinerea also strongly inhibited growth of Monilinia fructigena and Colletotrichum acutatum in vitro. Pathogenicity tests confirmed that none of the 13 isolates induced hypersensitive reactions on tobacco or caused rot in potato tissues. However, five isolates (T08/33, T08/48, T5/1, T14/8, and T14/1A) were pathogenic in the red onion scale assay. Biological sciences/Microbiology Biological sciences/Plant sciences gray mold antagonistic bacteria biocontrol Pantoea allii Pantoea agglomerans Pantoea ananatis Figures Figure 1 Introduction Poland is the largest producer of apples in Europe and the fourth largest in the world. However, producing high-quality fruit is often challenging. Storage diseases caused by pathogenic fungi are a major factor contributing to fruit quality deterioration. Some pathogens become harmful during the growing season, while others cause the most significant losses after harvest. The most important pathogens include Botrytis cinerea (gray mold), Phlyctema vagabunda syn. Neofabraea alba (bull’s eye rot), Glomerella acutata – anamorph: Colletotrichum acutatum sensu lato (bitter rot), Penicillium expansum (blue mold), and Monilinia fructigena (brown rot) 16 , 22 . B. cinerea (gray mold) can infect both flowers and fruit during the growing season. In cold storage, healthy fruit may become infected through contact with mycelium or spores. A typical symptom of this disease is the formation of pockets of decaying fruit. Once an apple is affected, all adjacent fruits are at risk of infection, making disease control highly important 19 , 49 . P. vagabunda is one of the most significant storage diseases of apples. The pathogen infects fruit in the second half of summer, typically beginning about two months before harvest. Infections are always latent, with the first symptoms appearing only during cold storage 34 . Bitter rot, caused by C. acutatum sensu lato, can develop even before harvest. The pathogen infects fruit from flowering until the end of the growing season. Apparently healthy apples may be harvested and placed in storage, but the first symptoms usually appear when the fruit reaches consumer maturity 10 , 42 . Another important pathogen is P. expansum (blue mold), which infects apples through wounds sustained during harvest or storage. The disease is most common in varieties with delicate skin that are particularly prone to mechanical damage. In addition, the pathogen produces patulin, a harmful mycotoxin, in infected fruit 17 , 32 . Monilinia fructigena , the causal agent of brown rot, can affect apples throughout the growing season. The main factors influencing its occurrence during fruit growth and ripening include insect damage (e.g., Cydia pomonella ) and hail injuries. Spores can also enter through lenticels. In cold storage, this disease often appears early 30 . Effective protection against storage diseases must be reliable, economically viable, and safe. In recent years, biological products have increasingly been incorporated into plant protection programs to reduce the risk of fungicide residues in crops. This trend has been reinforced by the gradual withdrawal of many active fungicide substances, which means that for some diseases, biological control methods may become the only viable option. An increasing number of plant protection products containing live microorganisms as active ingredients are appearing on the market, and research in this field continues 9 , 22 , 46 . Results The isolations from the phyllosphere and soil carried out allowed the collection of 2027 bacterial isolates. In the test regarding dual-plate confrontation assay (preliminary screening) pool of bacteria tested in the designated categories were distributed as follows: 1) 1,182 isolates inhibited growth of B. cinerea creating growth inhibition zone; 2) 496 isolates that he fungus did not grow on the surface of the bacterial growth; 3) 349 isolates that did not show any activity and the fungus grew in their environment without any restrictions. In general, there were 1,678 of those that showed some properties towards B. cinerea . In the research on the screening of bacterial activity on apple fruit against gray mold the experiments were carried out by inoculating fruits with two strains of B. cinerea (Iz1 and Iz2). All antagonistic isolates significantly protected the fruit against the development of gray mold after inoculation with strain Iz1. The following isolates caused the highest protection: T5/1 and T14/15 (effectiveness 77.5% and 77.2% respectively), as well as T16/8 (71.1%), T14/1A (66.2%) and M13/07 (62.4%). The remaining isolates reduced gray mold ranging from 23.8% to 43.7% (Table 2 ). Similarly, after inoculation with the Iz2 strain, all selected bacteria significantly reduced the occurrence of gray mold on apples. However, isolates T5/1, T14/1A, and T14/15 provided the most effective protection - effectiveness of 77.1%, 78.6%, and 75.7%, respectively. Suspensions 22ZK and 132M had the weakest effect (effectiveness 35.7% and 36.0% respectively), while the remaining isolates protected the fruit in the range from 46.0% to 69.4% (Table 2 ). Table 2 Screening on apple fruit against gray mold ( Botrytis cinerea ) Combination B. cinerea (Iz1) B. cinerea (Iz2) Control 31.1*± 0.7 f** 35.0 ± 6.4 c 22ZK 21.2 ± 6.0 de [31.8]*** 22.5 ± 1.7 b [35.7] 24PK 23.7 ± 5.8 e [23.8] 13.4 ± 2.6 ab [61,7] 131M 22.0 ± 2.3 e [29.3] 15.4 ± 0.9 ab [56.0] 132M 18.6 ± 3.3 cde [40.2] 22.4 ± 7.7 b [36.0] 133M 19.2 ± 7.2 cde [38.3] 18.9 ± 8.3 ab [46.0] M13/07 11.7 ± 1.2 abcd [62.4] 10.7 ± 3.7 ab [69.4] T08/33 17.5 ± 3.7 bcde [43.7] 11.3 ± 4.1 ab [67.7] T08/48 20.6 ± 4.6 de [33.7] 13.2 ± 4.6 ab [62.3] T5/1 7.0 ± 1.3 a [77.5] 8.0 ± 0.5 a [77.1] T14/1A 10.5 ± 1.6 abc [66.2] 7.5 ± 1.8 a [78.6] T14/8 20.1 ± 3.2 cde [35.5] 23.1 ± 2.6 b [34,0] T14/15 7.1 ± 1.9 a [77.2] 8.5 ± 1.4 a [75.7] T16/8 9.0 ± 2.2 ab [71.1] 11.3 ± 2.6 ab [67.7] * average percentage of infection from measurements for 3 apples and 4 necroses from each apple (total value from 12 measurements), ** The results were statistically developed using the variance analysis method on untransformed data; means within column followed by the same letter are not significantly different at P < 0.05 according to Newman–Keuls test (mean ± SE), *** efficiency in % (percentage of infection in relation to the control), Concentration of B. cinerea : 10 6 spores/ml and bacterial suspensions: 10 7 cfu/ml. In research on investigations of biotic relations between bacteria and pathogenic fungi (dual plate confrontation assay), selected bacteria most strongly limited the in vitro growth of: B. cinerea , M. fructigena and C. acutatum . The test carried out against gray mold ( B. cinerea strain Iz2) revealed that isolates 131M, T14/1A and T16/8 did not form any zones in the first test, and the pathogen did not grow on their surface. For isolates 22ZK, 24PK, 132M and 133M, the zones remained clear and the most extensive until the end of the assessment (14 days from application to the medium) and practically without noticeable changes (from 16.0 to 17.8 mm, depending on the date of the evaluation). However, the isolates: M13/07, T08/33, T08/48, T5/1, T14/8, T14/15, formed small zones ranging from 0.3 to 2 mm after 7 days. After the next 7 days, only M13/07 maintained a zone of 0.7 mm, and for the remaining five isolates, the mycelium growth of B. cinerea reached the edges of bacterial growth but did not grow on their surfaces (Table 3 ). Table 3 Biotic relationships between bacteria and selected pathogenic fungi occurring on apple trees Strain B. cinereal (Iz2) P. expansum (PEDJ8) M. fructigena (MFPD4) C. acutatum (CZA32) P. vagabunda (PNJ1) 7* 14* 7 14 7 14 7 14 14 22ZK 16.8 ± 0.1f** 16.5 ± 0.1 e 18.5 ± 0.1 b 17.7 ± 0.1 c 36.2 ± 1.0 f 36.2 ± 1.0 f 15.7 ± 0.1 k 5.5 ± 0.1 f 1.2 ± 0.1 b 24PK 17.7 ± 0.1 g 16.7 ± 0.1 f 19.1 ± 0.1 c 17.2 ± 0.1 b 35.1 ± 1.0 f 35.1 ± 1.0 f 15.2 ± 0.1 j 3.6 ± 0.1 d 2.1 ± 0.1 c 131M 0.0 ± 0.0# a 0.0 ± 0.0#a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.3 ± 0.1 a 0.0 ± 0.0 a 0.0 ± 0.0 a 132M 17.8 ± 0.1 g 16.0 ± 0.1 c 20.6 ± 0.1 d 19.7 ± 0.1 d 35.3 ± 1.0 f 35.3 ± 1.0 f 14.2 ± 0.1 i 3.1 ± 0.1 c 4.0 ± 0.1 f 133M 16.7 ± 0.1 f 16.2 ± 0.1 d 21.1 ± 0.1 e 20.3 ± 0.1 e 19.2 ± 1.0 d 19.2 ± 1.0 d 15.3 ± 0.1 j 3.8 ± 0.1 e 5.4 ± 0.1 h M13/07 0.9 ± 0.1 d 0.7 ± 0.1 b 0.0 ± 0.0# a 0.0 ± 0.0# a 3.0 ± 1.0 b 3.0 ± 1.0 b 4.3 ± 0.1 e 0.0 ± 0.0# a 2.0 ± 0.1 c T08/33 0.4 ± 0.1 b 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 1.0 ± 1.0 ab 0.0 ± 0.0# a 2.3 ± 0.1 c 0.0 ± 0.0# a 1.1 ± 0.1 b T08/48 0.6 ± 0.1 c 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 2.0 ± 1.0 b 0.0 ± 0.0# a 9.2 ± 0.1 f 0.0 ± 0.0# a 3.2 ± 0.1 e T5/1 2.0 ± 0.1 e 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 25.3 ± 1.0 e 25.3 ± 1.0 e 1.2 ± 0.1 b 1.2 ± 0.1 b 2.4 ± 0.1 d T14/1A 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 20.1 ± 1.0 d 5.0 ± 1.0 c 11.0 ± 0.1h 0.0 ± 0.1# a 1.3 ± 0.1 b T14/8 0.5 ± 0.1 c 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 2.0 ± 1.0 b 0.0 ± 0.0# a 2.7 ± 0.1 d 0.0 ± 0.0# a 2.5 ± 0.1 d T14/15 0.3 ± 0.1 b 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 1.0 ± 1.0 ab 0.0 ± 0.0# a 9.8 ± 0.1 g 0.0 ± 0.0# a 4.2 ± 0.1 g T16/8 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 0.0 ± 0.0# a 15.2 ± 1.0 c 5.0 ± 1.0 c 2.8 ± 0.1 d 0.0 ± 0.0# a 2.5 ± 0.1 d * time of measurement, ** inhibition zones of fungi growth (mm) caused by antagonistic bacteria on PDA medium, # not forming zones, mycelium did not cover the surface of the bacterial growth, statistical analysis was performed separately for each isolate and term; means with the same letters are not significantly different at P < 0.05 according to Newman-Keuls test (mean ± SE). For M. fructigena (strain MFPD4), in the first period (after 7 days) of observations, the largest zones were recorded for isolates 22ZK, 24PK, 132M, 133M, T5/1 T14/1A and T16/8 (from 15.2 to 36.2 mm). The remaining isolates ranged from 1.0 to 3.0 mm. Isolate 131M did not create growth restriction zones (Table 3 ). After the next 7 days, no changes were observed for isolates 22ZK, 24PK, 131M, 132M, 133M, M13/07 and T5/1. For isolates T14/1A and T16/8, a significant reduction in zones (up to 5 mm) was noted. In the case of the remaining isolates, the zones disappeared (Table 3 ). In the case of C. acutatum (strain CZA32), for isolates 22ZK, 24PK, 132M and 133M, after 7 days the zones ranged from 14.2 to 15.7 mm, but after another seven days they were significantly reduced and ranged from 3.1 to 5.5 mm. Isolate 131M formed a minimal 0.3 mm zone, which then disappeared after the next 7 days. Isolate T14/1A, although it formed a distinct zone (11 mm), disappeared after 14 days. The remaining isolates formed zones ranging in size from 2.3 mm to 9.8 mm, which, however, disappeared thoroughly after 14 days (Table 3 ). For P. expansum (strain PEDJ8), only four isolates: 22ZK, 24PK, 132M and 133M formed zones ranging from 17.2 to 21.1 mm depending on the assessment date. The remaining isolates did not limit the growth of the fungus on PDA medium (Table 3 ). Due to the slow growth of P. vagabunda (strain PNJ1), one reading was taken after 14 days, with only 131M not forming any zones, while the others ranged from 1.1 to 5.4 mm (Table 3 ). The 13 most active isolates were identified based on 16S rRNA sequence as well as on selected housekeeping marker genes. Eight isolates were classified to genus Pantoea ( P. agglomerans, P. allii, P. ananatis ), three to species Serratia inhibens , and one to Pseudomonas alvandae , Fig. 1 (Table 4 ). Table 4 List of the most efficient antagonistic and some of their characteristics determined in this study Strain Identity based on 16S rDNA (GenBank accession number) Identity based on other housekeeping genes sequences Sources of isolation selected isolates Test on onion scales HR Potato assay 22ZK Serratia inhibens ( PP660574) Serratia inhibens PV151383 ( infB ) PV151390 ( rpoB ) Apple rhizoplane cv. Antonovka nd - - 24PK Serratia inhibens ( PP660575) Serratia inhibens PV151389 ( rpoB ) As above nd - - 131M Pseudomonas laurylsulfativorans (PP664062) Pseudomonas laurylsulfatiphila PV151374 ( gyrB ) PV151375 ( rpoB ) Surface of apple leaves cv. Idared nd - - 132M Serratia inhibens (PP660576) Serratia inhibens PV151382 ( infB ) PV151388 ( rpoB ) As above nd - - 133M Pseudomonas alvandae (PP664061) - As above nd - - M13/07 Pantoea agglomerans (PP660862) Pantoea agglomerans PV151378 ( infB ) PV151381 ( rpoB ) Surface of raspberry leaves cv. Polka - - - T08/33 Pantoea allii (PP660869) Pantoea allii PV151377 ( infB ) PV151380 ( rpoB ) Surfaces of strawberry leaves cv. Onebor + - - T08/48 Pantoea allii (PP660868) Pantoea allii PV151376 ( infB ) PV151379 ( rpoB ) As above + - - T5/1 Pantoea ananatis (PP660863) Pantoea ananatis PX375375 ( gyrB ) PX375376 ( atpD ) PX375377 ( rpoB ) PX375378 ( infB ) As above + - - T14/1A Pantoea ananatis (PP660867) Pantoea ananatis PX375379 ( rpoB ) PX375380 ( infB ) As above + - - T14/15 Pantoea allii (PP660866) Pantoea allii PP399106 ( gyrB ) PP399107 ( atpD ) PP399108 ( rpoB ) PP399109 ( infB ) As above - - - T16/8 Pantoea pleuroti* (PP660864) Pantoea agglomerans PP399110 ( gyrB ) PP399111 ( atpD ) PP399112 ( rpoB ) PP399113 ( infB ) As above - - - T14/8 Pantoea ananatis (PP660865) Pantoea ananatis PV151384 ( infB ) PV151385 ( gyrB ) PV151386 ( atpD ) PV151387 ( rpoB ) As above + - - nd - not determined; + positive; – negative * not correct identification – 16S rRNA sequence resolution in this case was insufficient In the pathogenicity tests performed, none of the selected 13 isolates showed a positive reaction in hypersensitivity tests to tobacco and the ability to rot potato tissues. However, five isolates: T08/33, T08/48, T5/1, T14/8 and T14/1A were pathogenic in the test on red onion scales. Discussion In the present study, soil and epiphytic bacteria were isolated from various horticultural plants. These environmental niches are well known as sources of bacteria with potential for biological control 44 , 52 . The phyllosphere and soil have been recognized by many researchers as valuable environments not only for isolating fungal or yeast antagonists 21 , 8 but also bacterial antagonists 3 , 26 , 33 , 23 intended for controlling a broad spectrum of plant pathogens 14 . The microorganisms obtained in this study constituted the starting material for assessing their interactions with pathogenic fungi responsible for the most serious storage diseases of apples. Our work focused exclusively on bacteria with antagonistic potential, as the aim was to develop a bacteria-based product for practical use in reducing reliance on chemical pesticides. Moreover, the use of biopreparations helps prevent the development of fungicide resistance in plant pathogens 44 . Consequently, we excluded antagonistic yeasts and fungi from preliminary research, as their practical application in fruit production is limited by their sensitivity to fungicides. This sensitivity is particularly important when alternating biopreparations with chemical fungicides during the growing season. For instance, Kunz et al. 28 reported that in the case of BlossomProtect™ (based on Aureobasidium pullulans ), fungicides must not be applied on the same day or the following day after treatment with the biopreparation. Similar limitations apply to fungal antagonists, which can be inhibited by fungicides used against pathogens. The main potential obstacle for bacterial antagonists, by contrast, could be interactions with antibiotics. However, this issue is largely irrelevant, as the use of antibiotics in plant protection is currently prohibited in many countries, including all EU member states. Since our study involved screening a large number of bacterial isolates (over 2,000) from diverse environments, it was necessary to implement a selection scheme to gradually reduce their number. Therefore, the first stage of in vitro screening was limited to gray mold. This pathogen was chosen as the primary selection criterion because it is a widespread and serious disease of many crops, causing damage both during cultivation and post-harvest storage 12 , 55 . Another advantage was its rapid growth on PDA medium, which allowed for relatively quick assessment of results. The isolates that passed this stage were then tested on apple fruit against gray mold. Although labor-intensive and time-consuming, these tests enabled the reliable identification of 13 isolates that appeared most promising for practical use in apple orchards. In our view, relying solely on inhibition zone size in vitro is not always appropriate when selecting candidates for biological control, since some species (e.g., Pantoea agglomerans ) often produce small or no inhibition zones but are highly effective in in vivo tests 35 . In the study of biotic interactions on microbiological media, we assessed both the diameter of inhibition zones around bacterial colonies and the absence of fungal growth on bacterial surfaces. The former is typically associated with antibiotic production, a common trait among antagonists 47 , 44 , while the latter may indicate hyperparasitism, also described as a mechanism of bacterial antagonism 41 . Unlike traditional approaches, which prioritize microorganisms producing antifungal metabolites in vitro and reject those lacking antibiotic activity 2 , we applied a different selection method. While in vitro screening for antibiosis is still the most common first step in biocontrol research 39 , 44 , we emphasized results obtained in fruit assays as a more reliable criterion. Among the selected isolates, the largest group (eight isolates) consisted of epiphytic bacteria producing yellow pigment, representing various species of the genus Pantoea ( P. agglomerans, P. allii, P. ananatis ). These bacteria typically produced small inhibition zones or none at all, but prevented the overgrowth of B. cinerea or P. expansum on their colony surfaces. The fungistatic properties of Pantoea spp. are well documented in relation to vegetable pathogens 40 , 59 and fruit pathogens 5 , 52 , 36 . With respect to apples, Pantoea spp. have been reported to provide protection against storage diseases. For example, Nunes et al. 37,38 demonstrated nearly 100% effectiveness of these bacteria in preventing gray mold and blue mold on stored apples and pears. Our study showed that isolates of P. agglomerans , P. ananatis , and P. allii inhibited fungal growth only weakly or not at all on PDA medium. However, in apple assays, these isolates provided the best control of gray mold. Thus, we prioritized fruit assays over in vitro results. A notable observation was that none of the isolates allowed pathogenic fungi to grow on their surface, suggesting strong potential for use in storage environments, where pathogen–antagonist interactions occur mainly on wound surfaces caused by mechanical damage 39 . This observation is consistent with earlier studies showing that P. agglomerans isolates with weak or no antibiosis in vitro can still provide effective disease control in vivo—for example, against Stemphylium vesicarium on pear leaves 36 , Penicillium digitatum and P. italicum on citrus fruit 43 , and B. cinerea on grapevine leaves 52 . Another important species identified in our study was P. ananatis , previously reported as an endophyte with protective activity against Xanthomonas axonopodis pv. vesicatoria in peppers 20 . Importantly, while P. agglomerans and P. ananatis are well documented as biocontrol agents, no previous studies have described P. allii in this context 14 . The T14/15 isolate identified in our work, belonging to P. allii , appears promising for further development. Another bacterial group selected in this study belonged to the genus Pseudomonas. The antagonistic activity of Pseudomonas spp. against pathogenic fungi is widely recognized—for example, inhibition of B. cinerea conidial germination 53 or suppression of gray mold on lettuce and tomato in greenhouses 31 . Koomen and Jeffries 26 also identified Pseudomonas fluorescens as a promising postharvest biocontrol agent against Colletotrichum gloeosporioides on mango. Furthermore, P. fluorescens has been shown to inhibit Geotrichum candidum , the causal agent of carrot bitter rot 51 . In our study, we identified Pseudomonas laurylsulfatiphila and P. alvandae . The former did not typically form inhibition zones (except for small zones against C. acutatum ), but displayed distinctive UV fluorescence and prevented the growth of B. cinerea on its surface during initial assays. To date, no reports exist on the use of these species as biocontrol agents. Isolates of the genus Serratia were also selected because they produced extensive inhibition zones and demonstrated antagonistic activity against all tested pathogenic fungi. Serratia spp. are known for their rich secondary metabolism, producing various bioactive compounds such as the β-lactam antibiotic carbapenem and the antifungal metabolite oocydin 48 . Numerous studies have confirmed the antifungal activity of Serratia strains used as biocontrol agents 29 , 4 , 15 . However, their application is limited by safety concerns, as some isolates can be pathogenic to humans and other hosts 48 . Notably, S. inhibens , first detected on potato in Denmark, has been shown to strongly inhibit Rhizoctonia solani in vitro 18 . Overall, our study demonstrates the potential of several bacterial isolates for further research on plant–pathogen interactions and for postharvest applications where synthetic fungicides are restricted due to residue concerns. Importantly, this is the first report identifying Pantoea allii as a potential biocontrol agent. Material and Methods Isolation and storage of microorganisms Phyllosphere. Apparently healthy leaves from apple trees, strawberry, raspberry and grapes growing in the five plantations located in central Poland were collected 20 times during the 2017 and 2018 seasons (from April to September). Each sample, consisting of 10–20 leaves, was placed into 250 ml of sterile distilled water containing 0.02% Tween-20 in a 500 ml flask. It was then shaken on a reciprocal shaker at 150 rev./min for 30 minutes. Afterwards, 0.1 ml of the final wash was spread onto 2.3% nutrient agar medium (BD Difco™) supplemented with 5% sucrose (NAS). After 2 days of incubation at 25°C, morphologically distinct colonies were selected, purified on the same medium and used for further studies. The criteria used to select the bacterial isolates for the next stage were based on their ability to exhibit rapid growth and the visibility of their single colonies within a 24-48-hour time frame. From the microbial pool, only bacteria were selected. All selected isolates were incubated for 24 h on KingB medium 24 and then washed with PBS buffer and frozen in a mixture of PBS buffer (0.27% Na2HPO4, 0.04% NaH2PO4, 0.8% NaCl) and glycerol (4:1) at -80ºC. Soil. Soil samples were collected from 5 plantations of apple, strawberry and raspberry located in central Poland. Samples of 1 gram were collected twenty times during the seasons of 2017 and 2018. Each sample was placed in 100 ml of sterile water and shaken on a reciprocal shaker similarly (parameters) as in the case of leaf samples. Also, bacteria were isolated from the final wash and stored in glycerol at -80ºC in the same way as from leaves. Pathogens used in experiments In case of each pathogen, after washing the cultures out of the medium in a Petri dish, a 250 ml Erlenmeyer flask filled with 100 ml of suspension was placed on a shaker to break up spore clusters. After about 2 hours of shaking, the suspensions were standardised using a Bürker chamber (10 6 spores per millilitre). All strains of pathogenic fungi were multiplied on PDA (BD Difco™). From the mycelium, discs were cut with a cork-borer and placed in glycerol in 1.5 ml Eppendorf tubes. They were frozen at -80°C. Table 1 Pathogens, their source of isolation and suspension preparation Species Strain Host/ year of isolation Method of the conidial fungal spores inoculum preparation Botrytis cinerea Iz1 Iz2 decayed apple fruits cv. ‘Gala’/2018 decayed apple fruits cv. ‘Golden Delicious’ /2018 were obtained by washing the 14-day-old cultures growing on PDA medium with sterile distilled water Penicillium expansum PEDJ8 decayed apple fruits cv. ‘Red Jonaprince’ /2018 were obtained by washing the 7-day-old cultures growing on PDA medium with sterile distilled water Monilinia fructigena MFPD4 decayed apple fruits cv. ‘Piros’/2017 were obtained by washing the surface of the apples that were earlier inoculated. After 2 weeks from inoculation, the fruits were covered by sporodochia with conidial spores. Glomerella acutata (anamorph: Colletotrichum acutatum sensu lato) CZA32 decayed apple fruits cv. ‘Gala’/2015 were obtained by washing the 21-day-old cultures growing on PDA medium with sterile distilled water Phlyctema vagabunda PNJ1 decayed apple fruits cv. ‘Jonagold’/2015 were obtained by washing the 21-day-old cultures growing on V8* medium with sterile distilled water *composition of the medium: multi-vegetable juice 200 ml, calcium carbonate 3 g, technical agar BD Difco™ 20 g, distilled water 800 ml, pH of medium 5.6 Dual-plate confrontation assay for all isolates (preliminary screening) In the first stage, the antagonistic abilities of all 2027 collected bacterial isolates were determined in dual-plate confrontation assays against the gray mold ( B. cinerea ). To evaluate the interactions between the investigated microorganisms and the pathogen in culture, gray mold was co-cultured with each isolate on PDA (Difco). Petri dishes (containing PDA medium) were inoculated with 5 mm diameter discs with mycelium of B. cinerea that had been obtained (by cork-borer) from the margins of young colonies of the pathogen grown on the same medium. After 3 days, when the diameter of the fungus colony was about 1–2 cm, the tested bacteria were placed in a linear pattern at a distance of 50 mm from the fungus. For comparison purposes, the PDA plates with only the pathogenic fungus growing were used as a control. The cultures were incubated at room temperature (22ºC) for 14 days. The evaluation was carried out 7 and 14 days after the introduction of the bacteria. The diameters of the zone of fungal growth inhibition (bacterial activity) around the bacteria were measured and recorded. Three replicates were performed for each isolate. The assessment of the impact of microorganisms on fungi on PDA medium was estimated from 5 to 14 days, depending on the growth rate of the fungus. Isolates were divided into three categories: 1. Inhibiting the growth of B. cinerea (visible zone assessment in mm). 2. Not forming zones, the mycelium did not cover the surface of the bacterial growth. 3. Bacteria not showing any action (i.e. no zone, and fungus was growing on the surface of the bacteria). The microorganisms classified to the first two categories were considered as active isolates and were used in the following stages of the study, where they were tested in screening on apple fruit against gray mold. Testing of bacterial activity on apple fruit against gray mold ( B. cinerea ) The test was carried out only for those isolates that caused in vitro inhibition of B. cinerea growth, or the pathogen didn’t overgrow the tested bacterium (1,678 isolates). The experiments were carried out on Gala Must apple fruits that were collected in the Horticulture Experimental Orchard of the National Institute of Horticultural Research (InHort) in Dąbrowice from trees that were not protected by fungicides against storage diseases. The fruits were collected at the beginning of September in 2018 at the stage of harvest maturity (BBCH 87- fruit ripe for picking). Immediately after harvesting, the apples were placed in the storage chamber at 2°C and a relative humidity of 90–92%. After three months, when the apples reached consumption ripeness, the test was carried out. Apples were pre-cleaned by rinsing in water. After drying, the skin surface was sterilised by wiping with cotton soaked in 70% ethanol. On the perimeter in the central part of each fruit, four symmetrical wounds with a diameter of 5 mm and a depth of 3 mm were made with a cork-borer. Immediately afterwards, 30 µl of tested bacterial (or water in case of control) suspension was introduced into each wound (or sterile water in the case of control), and after 1 hour, an additional 30 µl of conidial spore suspension of the respective fungal isolate. Apples were stored for 4 days at room temperature (approx. 20°C) and relative humidity above 90%. Immediately after this time (4 days), the diameter of the rots was measured for each wound by making two cross measurements perpendicular to each other for each wound. Measurement values ​​were expressed in millimeters. Each isolate was tested on three apples, four replicates on each (12 lesions). The statistical analysis was calculated based on the average of all rots on each fruit (1 fruit = 1 replication). All tested bacterial suspensions at a concentration of 10 8 cfu/ml were used in the screening (1 and 2 groups). Over 4 tons of apples were used for the entire experiment. Efficacy in investigated combinations was expressed in comparison to the control. The experiment was repeated in the same way with the selected 13 most active isolates. Bacteria with the highest protective capabilities were compared for suspensions ten times lower, 10 7 cuf/ml, in the duplicate grey mould suppression. Investigation on biotic relations between bacteria and pathogenic fungi (dual plate confrontation assay) The experiment was conducted in the same manner as described in the dual-plate confrontation assay - preliminary screening. Other pathogenic apple fungi other than B. cinerea were included in the study. Antagonism of selected bacterial isolates was tested against: Penicillium expansum , Monilinia fructigena , Colletotrichum acutatum , Phlyctema vagabunda . Identification of bacterial isolates DNA-based identification of studied isolates From a 48 h culture of bacteria growing on NSA medium, DNA isolation was done with the Genomic Mini bacterial DNA isolation kit (A&A Biotechnology, Poland) according to the manufacturer's instructions. Sequence analysis of the 16S rRNA gene At the beginning, for the preliminary identification process of isolates, the 16S rRNA gene was amplified and sequenced. Amplification reactions were performed in a Biometra® T3000 thermal cycler by using fD1 and rP2 primers 56 and DreamTaq Green DNA polymerase (ThermoScientific, Vilnius, Lithuania) according to the conditions described in the original paper. The resulting amplification reaction product was separated on a 1.5% agarose gel in 0.5 x TBE buffer. To assess the size of the obtained product, 1 µl of O'Gene Ruler100 bp Plus DNA Ladder (ThermoFisherScientific, Waltham, MA, USA) was used. The product was sequenced at Genomed S.A. (Warsaw, Poland). The sequences obtained by automatic sequencing were assembled using the SeqMan program of the Lasergene software package (DNASTAR Inc., Madison, Wisconsin). The sequences of each isolate were compared to the sequences deposited in the Eztaxon database ( https://www.ezbiocloud.net/ ) 57 to find the closest relatives and classify the genera and higher taxa. Analysis of sequence of housekeeping genes fragments Additionally for four the most efficient strains, T16/8, T14/15, T5/1 and T14/8 and other strains of Pantoea genus sequence analysis of fragments of four housekeeping genes: atpD, gyrB, infB and rpoB were done. For this purpose, primers specific to the genes atpD, infB, gyrB 6 and rpoB 13 genes were used for the amplification. For other strains of Pantoea genus i.e. M13/07, T08/33, T08/48, T14/1A genes infB 6 and rpoB 13 were amplified. For bacteria classified as Pseudomonas sp. sequence analysis of fragments of two housekeeping genes: gyrB, and rpoB were done. For amplification the primers gyrB-F/gyrB-R 45 and LAPS/LAPS27 1 were used respectively. For bacteria of genus Serratia sequence analysis of fragments of two housekeeping genes were performed. For PCR the primers specific to the genes infB 6 and rpoB 45 genes were used. The sequences obtained by automatic sequencing were assembled using the SeqMan program of the Lasergene software package (DNASTAR Inc., Madison, Wisconsin) and compared to the sequences of the same genes of other species of Pantoea genus GenBank ( http://www.ncbi.nlm.nih.gov ) using the BLAST N program. For the phylogenetic analysis concatenated sequences of four genes of the most efficient strains were done (accession numbers are given in the section: Results and discussion, Table 4 ). The maximum likelihood (ML) tree were generated with the MEGA X software package 27 using the parameters and evolutionary Tamura-Nei model found as the best substitution model calculated automatically by program. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter = 0,4418)). The rate variation model allowed for some sites to be evolutionarily invariable ([+ I], 27,27% sites). Pathogenicity tests Hypersensitive reaction (HR) on tobacco plants. All selected 13 isolates were tested for their ability to induce HR on tobacco plants according to the method of Klement 25 . The water-bacterial suspension was prepared from a culture of each isolate cultivated on the King's B medium. The suspensions of tested isolates were injected with a hypodermic syringe into the mesophyll of a fully expanded tobacco leaf cv. Samsun. Induction of HR was observed within 24–48 h. The test was repeated for each isolate at least four times. Pectolytic activity. All the bacterial isolates were tested on slices of potato tubers cv. Irys. The tubers were first washed under tap water, air dried, then momentarily dipped into 96% ethanol, flamed and cut into 10-mm thick slices, which were then placed on wet filter paper in Petri dishes. Approximately two loops of each bacterial isolate grown on NAS medium were uniformly spread onto the upper surface of 20 slices. Development of rot on the slices was examined 24–48 h after incubation at 25°C. Test on red onion scales. Since some species of the identified bacteria are documented as onion pathogens in the literature 50 , 11 , a test on the red onion scales was carried out for strains of these species in accordance with Zhao et al. 58 , with modifications, fresh onion bulbs (cv. Karmen) were used for the test. Single onion scales were cut into fragments with sides about 2–3 cm long and placed on petri dishes with tissue paper soaked in sterile distilled water. A 50 µL (1 × 108 CFU/mL) of the tested bacteria suspension was introduced into a small puncture made with a sterile needle in the scale fragments. The sterile water was used as the negative control. The onion scales were incubated at room temperature. Strains that caused scale necrosis were considered pathogenic. The inoculated bacteria were re-isolated and confirmed from the symptomatic inoculated onion scales as described above. Four replicates per strain were used for one experiment, and the experiment was conducted twice. Statistical analysis Differences between means were assessed using the Newman-Keuls test at a significance level of 5%. Statistical calculations were performed for the experimental results presented in Tables 2 and 3 . Declarations Funding declaration The studies were performed in the frame of project BioSafeFood “Development of a technology for producing high-quality, consumer-safe fruit and vegetables using new biopreparations to protect crops against diseases”, co-financed by UE from the European Regional Development Fund under the Smart Growth Operational Programme 2014–2020, contract no. POIR.04.01.02-00-0100/17 − 00 NCBiR. (National Centre for Research and Development). Competing interests The authors declare no competing interests. Author Contribution A.M. designed the project, analyzed the data, performed experimental studies and wrote the manuscript. J.P. designed the project, analyzed the data, performed experimental studies and wrote the manuscript. H.G. analyzed the data, performed experimental studies and wrote the manuscript. M.K. performed the experimental part devoted to the identification of bacteria, wrote the manuscript. All authors reviewed the manuscript. Acknowledgement The studies were performed in the frame of project BioSafeFood “Development of a technology for producing high-quality, consumer-safe fruit and vegetables using new biopreparations to protect crops against diseases”, co-financed by UE from the European Regional Development Fund under the Smart Growth Operational Programme 2014-2020, contract no. POIR.04.01.02-00-0100/17-00 NCBiR. (National Centre for Research and Development).All authors are the inventors of Polish patent entitled ‘Strains of the genus Pantoea spp. and the use of strains of the genus Pantoea spp. in plant protection’ pat. no. 245234. Data Availability Identity based on 16S rDNA - GenBank accession numbersStrain 22ZK Serratia inhibens accession number PP660574Strain 24PK Serratia inhibens accession number PP660575Strain 131M Pseudomonas laurylsulfativorans accession number PP664062Strain 132M Serratia inhibens accession number PP660576Strain 133M Pseudomonas alvandae accession number PP664061Strain M13/07 Pantoea agglomerans accession number PP660862Strain T08/33 Pantoea allii accession number PP660869Strain T08/48 Pantoea allii accession number PP660868Strain T5/1 Pantoea ananatis accession number PP660863Strain T14/1A Pantoea ananatis accession number PP660867Strain T14/15 Pantoea allii accession number PP660866Strain T16/8 Pantoea pleuroti accession number PP660864Strain T14/8 Pantoea ananatis accession number PP660865 References Ait Tayeb, L. A., Ageron, E., Grimont, F. & Grimont, P. Molecular phylogeny of the genus Pseudomonas based on rpoB sequences, application for the identification of isolates. Res. 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Control . 164 , 104766. https://doi.org/10.1016/j.biocontrol.2021.104766 (2021). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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05:24:19","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173522,"visible":true,"origin":"","legend":"","description":"","filename":"60ea8f010a11439cb03d5247a4c81ecd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7696222/v1/f56a80208c1359d02d7f2ecc.xml"},{"id":95166951,"identity":"ff697989-aa54-4b80-bb26-1300a022897b","added_by":"auto","created_at":"2025-11-05 05:24:19","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192059,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7696222/v1/7f25270af582278c721ec2ca.html"},{"id":95166946,"identity":"3179ae08-3549-415c-994b-481ec17427d2","added_by":"auto","created_at":"2025-11-05 05:24:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3699118,"visible":true,"origin":"","legend":"\u003cp\u003eThe maximum likelihood tree of nucleotide sequences for the concatenated sequence data of \u003cem\u003eatpD\u003c/em\u003e, \u003cem\u003egyrB\u003c/em\u003e, \u003cem\u003einfB\u003c/em\u003e and \u003cem\u003erpoB\u003c/em\u003e genes of strains \u003cem\u003ePantoea\u003c/em\u003e \u003cem\u003ealii\u003c/em\u003e T14/15, \u003cem\u003ePantoea\u003c/em\u003e \u003cem\u003eagglomerans\u003c/em\u003e T16/8, \u003cem\u003ePantoea \u0026nbsp;ananatis \u003c/em\u003e14/8 and 5/1 and type strains of \u003cem\u003ePantoea\u003c/em\u003e spp. Bootstrap values (expressed as percentages of 500 replications) are indicated at each node. As an outgroup, the sequence of the same genes of \u003cem\u003eXanthomonas axonoposis\u003c/em\u003e pv. axonopodis LMG 982\u003csup\u003eT\u003c/sup\u003e was used\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7696222/v1/b993cf47b9bdeb513f9e4d3a.jpg"},{"id":101397760,"identity":"bfe2d6e8-8ab6-48a9-ab88-07fca7512914","added_by":"auto","created_at":"2026-01-29 09:36:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1799989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7696222/v1/27a6c302-c73a-4334-a091-bf6125977646.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening and identification of epiphytic and soil bacteria beneficial in the control of postharvest apple disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePoland is the largest producer of apples in Europe and the fourth largest in the world. However, producing high-quality fruit is often challenging. Storage diseases caused by pathogenic fungi are a major factor contributing to fruit quality deterioration. Some pathogens become harmful during the growing season, while others cause the most significant losses after harvest. The most important pathogens include \u003cem\u003eBotrytis cinerea\u003c/em\u003e (gray mold), \u003cem\u003ePhlyctema vagabunda\u003c/em\u003e syn. \u003cem\u003eNeofabraea alba\u003c/em\u003e (bull\u0026rsquo;s eye rot), \u003cem\u003eGlomerella acutata\u003c/em\u003e \u0026ndash; anamorph: \u003cem\u003eColletotrichum acutatum\u003c/em\u003e sensu lato (bitter rot), \u003cem\u003ePenicillium expansum\u003c/em\u003e (blue mold), and \u003cem\u003eMonilinia fructigena\u003c/em\u003e (brown rot)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e (gray mold) can infect both flowers and fruit during the growing season. In cold storage, healthy fruit may become infected through contact with mycelium or spores. A typical symptom of this disease is the formation of pockets of decaying fruit. Once an apple is affected, all adjacent fruits are at risk of infection, making disease control highly important\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eP. vagabunda\u003c/em\u003e is one of the most significant storage diseases of apples. The pathogen infects fruit in the second half of summer, typically beginning about two months before harvest. Infections are always latent, with the first symptoms appearing only during cold storage\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Bitter rot, caused by \u003cem\u003eC. acutatum\u003c/em\u003e sensu lato, can develop even before harvest. The pathogen infects fruit from flowering until the end of the growing season. Apparently healthy apples may be harvested and placed in storage, but the first symptoms usually appear when the fruit reaches consumer maturity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Another important pathogen is \u003cem\u003eP. expansum\u003c/em\u003e (blue mold), which infects apples through wounds sustained during harvest or storage. The disease is most common in varieties with delicate skin that are particularly prone to mechanical damage. In addition, the pathogen produces patulin, a harmful mycotoxin, in infected fruit\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eMonilinia fructigena\u003c/em\u003e, the causal agent of brown rot, can affect apples throughout the growing season. The main factors influencing its occurrence during fruit growth and ripening include insect damage (e.g., \u003cem\u003eCydia pomonella\u003c/em\u003e) and hail injuries. Spores can also enter through lenticels. In cold storage, this disease often appears early\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEffective protection against storage diseases must be reliable, economically viable, and safe. In recent years, biological products have increasingly been incorporated into plant protection programs to reduce the risk of fungicide residues in crops. This trend has been reinforced by the gradual withdrawal of many active fungicide substances, which means that for some diseases, biological control methods may become the only viable option. An increasing number of plant protection products containing live microorganisms as active ingredients are appearing on the market, and research in this field continues\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe isolations from the phyllosphere and soil carried out allowed the collection of 2027 bacterial isolates. In the test regarding dual-plate confrontation assay (preliminary screening) pool of bacteria tested in the designated categories were distributed as follows: 1) 1,182 isolates inhibited growth of \u003cem\u003eB. cinerea\u003c/em\u003e creating growth inhibition zone; 2) 496 isolates that he fungus did not grow on the surface of the bacterial growth; 3) 349 isolates that did not show any activity and the fungus grew in their environment without any restrictions. In general, there were 1,678 of those that showed some properties towards \u003cem\u003eB. cinerea\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn the research on the screening of bacterial activity on apple fruit against gray mold the experiments were carried out by inoculating fruits with two strains of \u003cem\u003eB. cinerea\u003c/em\u003e (Iz1 and Iz2). All antagonistic isolates significantly protected the fruit against the development of gray mold after inoculation with strain Iz1. The following isolates caused the highest protection: T5/1 and T14/15 (effectiveness 77.5% and 77.2% respectively), as well as T16/8 (71.1%), T14/1A (66.2%) and M13/07 (62.4%). The remaining isolates reduced gray mold ranging from 23.8% to 43.7% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, after inoculation with the Iz2 strain, all selected bacteria significantly reduced the occurrence of gray mold on apples. However, isolates T5/1, T14/1A, and T14/15 provided the most effective protection - effectiveness of 77.1%, 78.6%, and 75.7%, respectively. Suspensions 22ZK and 132M had the weakest effect (effectiveness 35.7% and 36.0% respectively), while the remaining isolates protected the fruit in the range from 46.0% to 69.4% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eScreening on apple fruit against gray mold (\u003cem\u003eBotrytis cinerea\u003c/em\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" 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colname=\"c3\"\u003e\u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 ab [56.0]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e132M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 cde [40.2]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7 b [36.0]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e133M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2 cde [38.3]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3 ab [46.0]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM13/07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 abcd [62.4]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 ab [69.4]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT08/33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 bcde [43.7]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 ab [67.7]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT08/48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 de [33.7]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 ab [62.3]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT5/1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 a [77.5]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 a [77.1]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/1A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 abc [66.2]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 a [78.6]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 cde [35.5]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 b [34,0]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 a [77.2]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 a [75.7]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT16/8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 ab [71.1]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 ab [67.7]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e* average percentage of infection from measurements for 3 apples and 4 necroses from each apple (total value from 12 measurements),\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e** The results were statistically developed using the variance analysis method on untransformed data; means within column followed by the same letter are not significantly different at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 according to Newman\u0026ndash;Keuls test (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE),\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e*** efficiency in % (percentage of infection in relation to the control),\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eConcentration of \u003cem\u003eB. cinerea\u003c/em\u003e: 10\u003csup\u003e6\u003c/sup\u003e spores/ml and bacterial suspensions: 10\u003csup\u003e7\u003c/sup\u003e cfu/ml.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn research on investigations of biotic relations between bacteria and pathogenic fungi (dual plate confrontation assay), selected bacteria most strongly limited the in vitro growth of: \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eM. fructigena\u003c/em\u003e and \u003cem\u003eC. acutatum\u003c/em\u003e. The test carried out against gray mold (\u003cem\u003eB. cinerea\u003c/em\u003e strain Iz2) revealed that isolates 131M, T14/1A and T16/8 did not form any zones in the first test, and the pathogen did not grow on their surface. For isolates 22ZK, 24PK, 132M and 133M, the zones remained clear and the most extensive until the end of the assessment (14 days from application to the medium) and practically without noticeable changes (from 16.0 to 17.8 mm, depending on the date of the evaluation). However, the isolates: M13/07, T08/33, T08/48, T5/1, T14/8, T14/15, formed small zones ranging from 0.3 to 2 mm after 7 days. After the next 7 days, only M13/07 maintained a zone of 0.7 mm, and for the remaining five isolates, the mycelium growth of \u003cem\u003eB. cinerea\u003c/em\u003e reached the edges of bacterial growth but did not grow on their surfaces (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBiotic relationships between bacteria and selected pathogenic fungi occurring on apple trees\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e\u003cem\u003eB. cinereal\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(Iz2)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP. expansum\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(PEDJ8)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e\u003cem\u003eM. fructigena\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(MFPD4)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cem\u003eC. acutatum\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(CZA32)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cem\u003eP. vagabunda\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(PNJ1)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22ZK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1f**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 k\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24PK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e35.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e35.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 j\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e131M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0#a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e132M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e35.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e35.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 i\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 f\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e133M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e19.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 j\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 h\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM13/07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT08/33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT08/48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT5/1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/1A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT16/8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0# a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 d\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e* time of measurement,\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e** inhibition zones of fungi growth (mm) caused by antagonistic bacteria on PDA medium,\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e# not forming zones, mycelium did not cover the surface of the bacterial growth,\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003estatistical analysis was performed separately for each isolate and term; means with the same letters are not significantly different at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 according to Newman-Keuls test (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor \u003cem\u003eM. fructigena\u003c/em\u003e (strain MFPD4), in the first period (after 7 days) of observations, the largest zones were recorded for isolates 22ZK, 24PK, 132M, 133M, T5/1 T14/1A and T16/8 (from 15.2 to 36.2 mm). The remaining isolates ranged from 1.0 to 3.0 mm. Isolate 131M did not create growth restriction zones (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After the next 7 days, no changes were observed for isolates 22ZK, 24PK, 131M, 132M, 133M, M13/07 and T5/1. For isolates T14/1A and T16/8, a significant reduction in zones (up to 5 mm) was noted. In the case of the remaining isolates, the zones disappeared (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the case of \u003cem\u003eC. acutatum\u003c/em\u003e (strain CZA32), for isolates 22ZK, 24PK, 132M and 133M, after 7 days the zones ranged from 14.2 to 15.7 mm, but after another seven days they were significantly reduced and ranged from 3.1 to 5.5 mm. Isolate 131M formed a minimal 0.3 mm zone, which then disappeared after the next 7 days. Isolate T14/1A, although it formed a distinct zone (11 mm), disappeared after 14 days. The remaining isolates formed zones ranging in size from 2.3 mm to 9.8 mm, which, however, disappeared thoroughly after 14 days (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor \u003cem\u003eP. expansum\u003c/em\u003e (strain PEDJ8), only four isolates: 22ZK, 24PK, 132M and 133M formed zones ranging from 17.2 to 21.1 mm depending on the assessment date. The remaining isolates did not limit the growth of the fungus on PDA medium (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDue to the slow growth of \u003cem\u003eP. vagabunda\u003c/em\u003e (strain PNJ1), one reading was taken after 14 days, with only 131M not forming any zones, while the others ranged from 1.1 to 5.4 mm (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe 13 most active isolates were identified based on 16S rRNA sequence as well as on selected housekeeping marker genes. Eight isolates were classified to genus Pantoea (\u003cem\u003eP. agglomerans, P. allii, P. ananatis\u003c/em\u003e), three to species \u003cem\u003eSerratia inhibens\u003c/em\u003e, and one to \u003cem\u003ePseudomonas alvandae\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of the most efficient antagonistic and some of their characteristics determined in this study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIdentity based on 16S rDNA (GenBank accession number)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIdentity based on other housekeeping genes sequences\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSources of isolation selected isolates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTest on onion scales\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePotato assay\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22ZK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eSerratia inhibens\u003c/em\u003e \u003cb\u003e(\u003c/b\u003ePP660574)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eSerratia inhibens\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePV151383 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151390 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eApple rhizoplane cv. Antonovka\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24PK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eSerratia inhibens\u003c/em\u003e \u003cb\u003e(\u003c/b\u003ePP660575)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eSerratia inhibens\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePV151389 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e131M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas laurylsulfativorans\u003c/em\u003e (PP664062)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas laurylsulfatiphila\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePV151374 (\u003cem\u003egyrB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151375 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSurface of apple leaves cv. Idared\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e132M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eSerratia inhibens\u003c/em\u003e (PP660576)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eSerratia inhibens\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePV151382 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151388 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e133M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas alvandae\u003c/em\u003e (PP664061)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003end\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM13/07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePantoea agglomerans\u003c/em\u003e (PP660862)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePantoea agglomerans\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePV151378 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151381 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSurface of raspberry leaves cv. Polka\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT08/33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePantoea allii\u003c/em\u003e (PP660869)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePantoea allii\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePV151377 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151380 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSurfaces of strawberry leaves cv. Onebor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT08/48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePantoea allii\u003c/em\u003e (PP660868)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePantoea allii\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePV151376 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151379 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT5/1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePantoea ananatis\u003c/em\u003e (PP660863)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePantoea ananatis\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePX375375 (\u003cem\u003egyrB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePX375376 (\u003cem\u003eatpD\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePX375377 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePX375378 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/1A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePantoea ananatis\u003c/em\u003e (PP660867)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePantoea ananatis\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePX375379 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePX375380 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePantoea allii\u003c/em\u003e (PP660866)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePantoea allii\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePP399106 (\u003cem\u003egyrB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePP399107 (\u003cem\u003eatpD\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePP399108 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePP399109 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT16/8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePantoea pleuroti*\u003c/em\u003e (PP660864)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePantoea agglomerans\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePP399110 (\u003cem\u003egyrB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePP399111 (\u003cem\u003eatpD\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePP399112 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePP399113 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14/8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePantoea ananatis\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(PP660865)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePantoea ananatis\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePV151384 (\u003cem\u003einfB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151385 (\u003cem\u003egyrB\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151386 (\u003cem\u003eatpD\u003c/em\u003e)\u003c/p\u003e\u003cp\u003ePV151387 (\u003cem\u003erpoB\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAs above\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003end - not determined; + positive; \u0026ndash; negative\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e* not correct identification \u0026ndash; 16S rRNA sequence resolution in this case was insufficient\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the pathogenicity tests performed, none of the selected 13 isolates showed a positive reaction in hypersensitivity tests to tobacco and the ability to rot potato tissues. However, five isolates: T08/33, T08/48, T5/1, T14/8 and T14/1A were pathogenic in the test on red onion scales.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, soil and epiphytic bacteria were isolated from various horticultural plants. These environmental niches are well known as sources of bacteria with potential for biological control\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The phyllosphere and soil have been recognized by many researchers as valuable environments not only for isolating fungal or yeast antagonists\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e but also bacterial antagonists\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e intended for controlling a broad spectrum of plant pathogens\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe microorganisms obtained in this study constituted the starting material for assessing their interactions with pathogenic fungi responsible for the most serious storage diseases of apples. Our work focused exclusively on bacteria with antagonistic potential, as the aim was to develop a bacteria-based product for practical use in reducing reliance on chemical pesticides. Moreover, the use of biopreparations helps prevent the development of fungicide resistance in plant pathogens\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Consequently, we excluded antagonistic yeasts and fungi from preliminary research, as their practical application in fruit production is limited by their sensitivity to fungicides. This sensitivity is particularly important when alternating biopreparations with chemical fungicides during the growing season. For instance, Kunz et al.\u003csup\u003e28\u003c/sup\u003e reported that in the case of BlossomProtect\u0026trade; (based on \u003cem\u003eAureobasidium pullulans\u003c/em\u003e), fungicides must not be applied on the same day or the following day after treatment with the biopreparation. Similar limitations apply to fungal antagonists, which can be inhibited by fungicides used against pathogens. The main potential obstacle for bacterial antagonists, by contrast, could be interactions with antibiotics. However, this issue is largely irrelevant, as the use of antibiotics in plant protection is currently prohibited in many countries, including all EU member states.\u003c/p\u003e\u003cp\u003eSince our study involved screening a large number of bacterial isolates (over 2,000) from diverse environments, it was necessary to implement a selection scheme to gradually reduce their number. Therefore, the first stage of in vitro screening was limited to gray mold. This pathogen was chosen as the primary selection criterion because it is a widespread and serious disease of many crops, causing damage both during cultivation and post-harvest storage\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Another advantage was its rapid growth on PDA medium, which allowed for relatively quick assessment of results.\u003c/p\u003e\u003cp\u003eThe isolates that passed this stage were then tested on apple fruit against gray mold. Although labor-intensive and time-consuming, these tests enabled the reliable identification of 13 isolates that appeared most promising for practical use in apple orchards. In our view, relying solely on inhibition zone size in vitro is not always appropriate when selecting candidates for biological control, since some species (e.g., \u003cem\u003ePantoea agglomerans\u003c/em\u003e) often produce small or no inhibition zones but are highly effective in in vivo tests\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn the study of biotic interactions on microbiological media, we assessed both the diameter of inhibition zones around bacterial colonies and the absence of fungal growth on bacterial surfaces. The former is typically associated with antibiotic production, a common trait among antagonists\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, while the latter may indicate hyperparasitism, also described as a mechanism of bacterial antagonism\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUnlike traditional approaches, which prioritize microorganisms producing antifungal metabolites in vitro and reject those lacking antibiotic activity\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, we applied a different selection method. While in vitro screening for antibiosis is still the most common first step in biocontrol research\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, we emphasized results obtained in fruit assays as a more reliable criterion.\u003c/p\u003e\u003cp\u003eAmong the selected isolates, the largest group (eight isolates) consisted of epiphytic bacteria producing yellow pigment, representing various species of the genus Pantoea (\u003cem\u003eP. agglomerans, P. allii, P. ananatis\u003c/em\u003e). These bacteria typically produced small inhibition zones or none at all, but prevented the overgrowth of \u003cem\u003eB. cinerea\u003c/em\u003e or \u003cem\u003eP. expansum\u003c/em\u003e on their colony surfaces. The fungistatic properties of \u003cem\u003ePantoea\u003c/em\u003e spp. are well documented in relation to vegetable pathogens\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and fruit pathogens\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. With respect to apples, \u003cem\u003ePantoea\u003c/em\u003e spp. have been reported to provide protection against storage diseases. For example, Nunes et al.\u003csup\u003e37,38\u003c/sup\u003e demonstrated nearly 100% effectiveness of these bacteria in preventing gray mold and blue mold on stored apples and pears.\u003c/p\u003e\u003cp\u003eOur study showed that isolates of \u003cem\u003eP. agglomerans\u003c/em\u003e, \u003cem\u003eP. ananatis\u003c/em\u003e, and \u003cem\u003eP. allii\u003c/em\u003e inhibited fungal growth only weakly or not at all on PDA medium. However, in apple assays, these isolates provided the best control of gray mold. Thus, we prioritized fruit assays over in vitro results. A notable observation was that none of the isolates allowed pathogenic fungi to grow on their surface, suggesting strong potential for use in storage environments, where pathogen\u0026ndash;antagonist interactions occur mainly on wound surfaces caused by mechanical damage\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This observation is consistent with earlier studies showing that \u003cem\u003eP. agglomerans\u003c/em\u003e isolates with weak or no antibiosis in vitro can still provide effective disease control in vivo\u0026mdash;for example, against \u003cem\u003eStemphylium vesicarium\u003c/em\u003e on pear leaves\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ePenicillium digitatum\u003c/em\u003e and \u003cem\u003eP. italicum\u003c/em\u003e on citrus fruit\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and B. \u003cem\u003ecinerea\u003c/em\u003e on grapevine leaves\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Another important species identified in our study was \u003cem\u003eP. ananatis\u003c/em\u003e, previously reported as an endophyte with protective activity against \u003cem\u003eXanthomonas axonopodis\u003c/em\u003e pv. \u003cem\u003evesicatoria\u003c/em\u003e in peppers\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Importantly, while \u003cem\u003eP. agglomerans\u003c/em\u003e and \u003cem\u003eP. ananatis\u003c/em\u003e are well documented as biocontrol agents, no previous studies have described \u003cem\u003eP. allii\u003c/em\u003e in this context\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The T14/15 isolate identified in our work, belonging to \u003cem\u003eP. allii\u003c/em\u003e, appears promising for further development.\u003c/p\u003e\u003cp\u003eAnother bacterial group selected in this study belonged to the genus Pseudomonas. The antagonistic activity of \u003cem\u003ePseudomonas\u003c/em\u003e spp. against pathogenic fungi is widely recognized\u0026mdash;for example, inhibition of \u003cem\u003eB. cinerea\u003c/em\u003e conidial germination\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e or suppression of gray mold on lettuce and tomato in greenhouses\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Koomen and Jeffries\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e also identified \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e as a promising postharvest biocontrol agent against \u003cem\u003eColletotrichum gloeosporioides\u003c/em\u003e on mango. Furthermore, \u003cem\u003eP. fluorescens\u003c/em\u003e has been shown to inhibit \u003cem\u003eGeotrichum candidum\u003c/em\u003e, the causal agent of carrot bitter rot\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In our study, we identified \u003cem\u003ePseudomonas laurylsulfatiphila\u003c/em\u003e and \u003cem\u003eP. alvandae\u003c/em\u003e. The former did not typically form inhibition zones (except for small zones against \u003cem\u003eC. acutatum\u003c/em\u003e), but displayed distinctive UV fluorescence and prevented the growth of \u003cem\u003eB. cinerea\u003c/em\u003e on its surface during initial assays. To date, no reports exist on the use of these species as biocontrol agents.\u003c/p\u003e\u003cp\u003eIsolates of the genus Serratia were also selected because they produced extensive inhibition zones and demonstrated antagonistic activity against all tested pathogenic fungi. \u003cem\u003eSerratia\u003c/em\u003e spp. are known for their rich secondary metabolism, producing various bioactive compounds such as the β-lactam antibiotic carbapenem and the antifungal metabolite oocydin\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Numerous studies have confirmed the antifungal activity of Serratia strains used as biocontrol agents\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, their application is limited by safety concerns, as some isolates can be pathogenic to humans and other hosts\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Notably, \u003cem\u003eS. inhibens\u003c/em\u003e, first detected on potato in Denmark, has been shown to strongly inhibit \u003cem\u003eRhizoctonia solani\u003c/em\u003e in vitro\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOverall, our study demonstrates the potential of several bacterial isolates for further research on plant\u0026ndash;pathogen interactions and for postharvest applications where synthetic fungicides are restricted due to residue concerns. Importantly, this is the first report identifying \u003cem\u003ePantoea allii\u003c/em\u003e as a potential biocontrol agent.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eIsolation and storage of microorganisms\u003c/p\u003e\u003cp\u003ePhyllosphere. Apparently healthy leaves from apple trees, strawberry, raspberry and grapes growing in the five plantations located in central Poland were collected 20 times during the 2017 and 2018 seasons (from April to September). Each sample, consisting of 10\u0026ndash;20 leaves, was placed into 250 ml of sterile distilled water containing 0.02% Tween-20 in a 500 ml flask. It was then shaken on a reciprocal shaker at 150 rev./min for 30 minutes. Afterwards, 0.1 ml of the final wash was spread onto 2.3% nutrient agar medium (BD Difco\u0026trade;) supplemented with 5% sucrose (NAS). After 2 days of incubation at 25\u0026deg;C, morphologically distinct colonies were selected, purified on the same medium and used for further studies. The criteria used to select the bacterial isolates for the next stage were based on their ability to exhibit rapid growth and the visibility of their single colonies within a 24-48-hour time frame. From the microbial pool, only bacteria were selected.\u003c/p\u003e\u003cp\u003eAll selected isolates were incubated for 24 h on KingB medium\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and then washed with PBS buffer and frozen in a mixture of PBS buffer (0.27% Na2HPO4, 0.04% NaH2PO4, 0.8% NaCl) and glycerol (4:1) at -80\u0026ordm;C.\u003c/p\u003e\u003cp\u003eSoil. Soil samples were collected from 5 plantations of apple, strawberry and raspberry located in central Poland. Samples of 1 gram were collected twenty times during the seasons of 2017 and 2018. Each sample was placed in 100 ml of sterile water and shaken on a reciprocal shaker similarly (parameters) as in the case of leaf samples. Also, bacteria were isolated from the final wash and stored in glycerol at -80\u0026ordm;C in the same way as from leaves.\u003c/p\u003e\u003cp\u003ePathogens used in experiments\u003c/p\u003e\u003cp\u003eIn case of each pathogen, after washing the cultures out of the medium in a Petri dish, a 250 ml Erlenmeyer flask filled with 100 ml of suspension was placed on a shaker to break up spore clusters. After about 2 hours of shaking, the suspensions were standardised using a B\u0026uuml;rker chamber (10\u003csup\u003e6\u003c/sup\u003e spores per millilitre).\u003c/p\u003e\u003cp\u003eAll strains of pathogenic fungi were multiplied on PDA (BD Difco\u0026trade;). From the mycelium, discs were cut with a cork-borer and placed in glycerol in 1.5 ml Eppendorf tubes. They were frozen at -80\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePathogens, their source of isolation and suspension preparation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHost/\u003c/p\u003e\u003cp\u003eyear of isolation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMethod of the conidial fungal spores inoculum preparation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBotrytis cinerea\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIz1\u003c/p\u003e\u003cp\u003eIz2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edecayed apple fruits cv. \u0026lsquo;Gala\u0026rsquo;/2018\u003c/p\u003e\u003cp\u003edecayed apple fruits cv. \u0026lsquo;Golden Delicious\u0026rsquo; /2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ewere obtained by washing the 14-day-old cultures growing on PDA medium with sterile distilled water\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePenicillium expansum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePEDJ8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edecayed apple fruits cv. \u0026lsquo;Red Jonaprince\u0026rsquo; /2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ewere obtained by washing the 7-day-old cultures growing on PDA medium with sterile distilled water\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMonilinia fructigena\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMFPD4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edecayed apple fruits cv. \u0026lsquo;Piros\u0026rsquo;/2017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ewere obtained by washing the surface of the apples that were earlier inoculated. After 2 weeks from inoculation, the fruits were covered by sporodochia with conidial spores.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGlomerella acutata\u003c/em\u003e (anamorph: \u003cem\u003eColletotrichum acutatum\u003c/em\u003e sensu lato)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCZA32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edecayed apple fruits cv. \u0026lsquo;Gala\u0026rsquo;/2015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ewere obtained by washing the 21-day-old cultures growing on PDA medium with sterile distilled water\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePhlyctema vagabunda\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePNJ1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edecayed apple fruits cv. \u0026lsquo;Jonagold\u0026rsquo;/2015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ewere obtained by washing the 21-day-old cultures growing on V8* medium with sterile distilled water\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e*composition of the medium: multi-vegetable juice 200 ml, calcium carbonate 3 g, technical agar BD Difco\u0026trade; 20 g, distilled water 800 ml, pH of medium 5.6\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDual-plate confrontation assay for all isolates (preliminary screening)\u003c/p\u003e\u003cp\u003eIn the first stage, the antagonistic abilities of all 2027 collected bacterial isolates were determined in dual-plate confrontation assays against the gray mold (\u003cem\u003eB. cinerea\u003c/em\u003e). To evaluate the interactions between the investigated microorganisms and the pathogen in culture, gray mold was co-cultured with each isolate on PDA (Difco).\u003c/p\u003e\u003cp\u003ePetri dishes (containing PDA medium) were inoculated with 5 mm diameter discs with mycelium of \u003cem\u003eB. cinerea\u003c/em\u003e that had been obtained (by cork-borer) from the margins of young colonies of the pathogen grown on the same medium. After 3 days, when the diameter of the fungus colony was about 1\u0026ndash;2 cm, the tested bacteria were placed in a linear pattern at a distance of 50 mm from the fungus. For comparison purposes, the PDA plates with only the pathogenic fungus growing were used as a control. The cultures were incubated at room temperature (22\u0026ordm;C) for 14 days. The evaluation was carried out 7 and 14 days after the introduction of the bacteria. The diameters of the zone of fungal growth inhibition (bacterial activity) around the bacteria were measured and recorded. Three replicates were performed for each isolate.\u003c/p\u003e\u003cp\u003eThe assessment of the impact of microorganisms on fungi on PDA medium was estimated from 5 to 14 days, depending on the growth rate of the fungus.\u003c/p\u003e\u003cp\u003eIsolates were divided into three categories:\u003c/p\u003e\u003cp\u003e1. Inhibiting the growth of B. cinerea (visible zone assessment in mm).\u003c/p\u003e\u003cp\u003e2. Not forming zones, the mycelium did not cover the surface of the bacterial growth.\u003c/p\u003e\u003cp\u003e3. Bacteria not showing any action (i.e. no zone, and fungus was growing on the surface of the bacteria).\u003c/p\u003e\u003cp\u003eThe microorganisms classified to the first two categories were considered as active isolates and were used in the following stages of the study, where they were tested in screening on apple fruit against gray mold.\u003c/p\u003e\u003cp\u003eTesting of bacterial activity on apple fruit against gray mold (\u003cem\u003eB. cinerea\u003c/em\u003e)\u003c/p\u003e\u003cp\u003eThe test was carried out only for those isolates that caused in vitro inhibition of \u003cem\u003eB. cinerea\u003c/em\u003e growth, or the pathogen didn\u0026rsquo;t overgrow the tested bacterium (1,678 isolates).\u003c/p\u003e\u003cp\u003eThe experiments were carried out on Gala Must apple fruits that were collected in the Horticulture Experimental Orchard of the National Institute of Horticultural Research (InHort) in Dąbrowice from trees that were not protected by fungicides against storage diseases. The fruits were collected at the beginning of September in 2018 at the stage of harvest maturity (BBCH 87- fruit ripe for picking). Immediately after harvesting, the apples were placed in the storage chamber at 2\u0026deg;C and a relative humidity of 90\u0026ndash;92%. After three months, when the apples reached consumption ripeness, the test was carried out.\u003c/p\u003e\u003cp\u003eApples were pre-cleaned by rinsing in water. After drying, the skin surface was sterilised by wiping with cotton soaked in 70% ethanol. On the perimeter in the central part of each fruit, four symmetrical wounds with a diameter of 5 mm and a depth of 3 mm were made with a cork-borer. Immediately afterwards, 30 \u0026micro;l of tested bacterial (or water in case of control) suspension was introduced into each wound (or sterile water in the case of control), and after 1 hour, an additional 30 \u0026micro;l of conidial spore suspension of the respective fungal isolate. Apples were stored for 4 days at room temperature (approx. 20\u0026deg;C) and relative humidity above 90%. Immediately after this time (4 days), the diameter of the rots was measured for each wound by making two cross measurements perpendicular to each other for each wound. Measurement values ​​were expressed in millimeters. Each isolate was tested on three apples, four replicates on each (12 lesions). The statistical analysis was calculated based on the average of all rots on each fruit (1 fruit\u0026thinsp;=\u0026thinsp;1 replication). All tested bacterial suspensions at a concentration of 10\u003csup\u003e8\u003c/sup\u003e cfu/ml were used in the screening (1 and 2 groups). Over 4 tons of apples were used for the entire experiment.\u003c/p\u003e\u003cp\u003eEfficacy in investigated combinations was expressed in comparison to the control. The experiment was repeated in the same way with the selected 13 most active isolates. Bacteria with the highest protective capabilities were compared for suspensions ten times lower, 10\u003csup\u003e7\u003c/sup\u003e cuf/ml, in the duplicate grey mould suppression.\u003c/p\u003e\u003cp\u003eInvestigation on biotic relations between bacteria and pathogenic fungi (dual plate confrontation assay)\u003c/p\u003e\u003cp\u003eThe experiment was conducted in the same manner as described in the dual-plate confrontation assay - preliminary screening. Other pathogenic apple fungi other than \u003cem\u003eB. cinerea\u003c/em\u003e were included in the study. Antagonism of selected bacterial isolates was tested against: \u003cem\u003ePenicillium expansum\u003c/em\u003e, \u003cem\u003eMonilinia fructigena\u003c/em\u003e, \u003cem\u003eColletotrichum acutatum\u003c/em\u003e, \u003cem\u003ePhlyctema vagabunda\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIdentification of bacterial isolates\u003c/p\u003e\u003cp\u003eDNA-based identification of studied isolates\u003c/p\u003e\u003cp\u003eFrom a 48 h culture of bacteria growing on NSA medium, DNA isolation was done with the Genomic Mini bacterial DNA isolation kit (A\u0026amp;A Biotechnology, Poland) according to the manufacturer's instructions.\u003c/p\u003e\u003cp\u003eSequence analysis of the 16S rRNA gene\u003c/p\u003e\u003cp\u003eAt the beginning, for the preliminary identification process of isolates, the 16S rRNA gene was amplified and sequenced. Amplification reactions were performed in a Biometra\u0026reg; T3000 thermal cycler by using fD1 and rP2 primers\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e and DreamTaq Green DNA polymerase (ThermoScientific, Vilnius, Lithuania) according to the conditions described in the original paper. The resulting amplification reaction product was separated on a 1.5% agarose gel in 0.5 x TBE buffer. To assess the size of the obtained product, 1 \u0026micro;l of O'Gene Ruler100 bp Plus DNA Ladder (ThermoFisherScientific, Waltham, MA, USA) was used. The product was sequenced at Genomed S.A. (Warsaw, Poland). The sequences obtained by automatic sequencing were assembled using the SeqMan program of the Lasergene software package (DNASTAR Inc., Madison, Wisconsin). The sequences of each isolate were compared to the sequences deposited in the Eztaxon database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e57\u003c/sup\u003e to find the closest relatives and classify the genera and higher taxa.\u003c/p\u003e\u003cp\u003eAnalysis of sequence of housekeeping genes fragments\u003c/p\u003e\u003cp\u003eAdditionally for four the most efficient strains, T16/8, T14/15, T5/1 and T14/8 and other strains of Pantoea genus sequence analysis of fragments of four housekeeping genes: atpD, gyrB, infB and rpoB were done. For this purpose, primers specific to the genes atpD, infB, gyrB\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and rpoB\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e genes were used for the amplification. For other strains of Pantoea genus i.e. M13/07, T08/33, T08/48, T14/1A genes infB\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and rpoB\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e were amplified. For bacteria classified as Pseudomonas sp. sequence analysis of fragments of two housekeeping genes: gyrB, and rpoB were done. For amplification the primers gyrB-F/gyrB-R\u003csup\u003e45\u003c/sup\u003e and LAPS/LAPS27\u003csup\u003e1\u003c/sup\u003e were used respectively. For bacteria of genus Serratia sequence analysis of fragments of two housekeeping genes were performed. For PCR the primers specific to the genes infB\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and rpoB\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e genes were used. The sequences obtained by automatic sequencing were assembled using the SeqMan program of the Lasergene software package (DNASTAR Inc., Madison, Wisconsin) and compared to the sequences of the same genes of other species of Pantoea genus GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using the BLAST N program. For the phylogenetic analysis concatenated sequences of four genes of the most efficient strains were done (accession numbers are given in the section: Results and discussion, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The maximum likelihood (ML) tree were generated with the MEGA X software package\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e using the parameters and evolutionary Tamura-Nei model found as the best substitution model calculated automatically by program. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+\u0026thinsp;G, parameter\u0026thinsp;=\u0026thinsp;0,4418)). The rate variation model allowed for some sites to be evolutionarily invariable ([+\u0026thinsp;I], 27,27% sites).\u003c/p\u003e\u003cp\u003ePathogenicity tests\u003c/p\u003e\u003cp\u003eHypersensitive reaction (HR) on tobacco plants. All selected 13 isolates were tested for their ability to induce HR on tobacco plants according to the method of Klement\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The water-bacterial suspension was prepared from a culture of each isolate cultivated on the King's B medium. The suspensions of tested isolates were injected with a hypodermic syringe into the mesophyll of a fully expanded tobacco leaf cv. Samsun. Induction of HR was observed within 24\u0026ndash;48 h. The test was repeated for each isolate at least four times.\u003c/p\u003e\u003cp\u003ePectolytic activity. All the bacterial isolates were tested on slices of potato tubers cv. Irys. The tubers were first washed under tap water, air dried, then momentarily dipped into 96% ethanol, flamed and cut into 10-mm thick slices, which were then placed on wet filter paper in Petri dishes. Approximately two loops of each bacterial isolate grown on NAS medium were uniformly spread onto the upper surface of 20 slices. Development of rot on the slices was examined 24\u0026ndash;48 h after incubation at 25\u0026deg;C.\u003c/p\u003e\u003cp\u003eTest on red onion scales. Since some species of the identified bacteria are documented as onion pathogens in the literature\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, a test on the red onion scales was carried out for strains of these species in accordance with Zhao et al.\u003csup\u003e58\u003c/sup\u003e, with modifications, fresh onion bulbs (cv. Karmen) were used for the test. Single onion scales were cut into fragments with sides about 2\u0026ndash;3 cm long and placed on petri dishes with tissue paper soaked in sterile distilled water. A 50 \u0026micro;L (1 \u0026times; 108 CFU/mL) of the tested bacteria suspension was introduced into a small puncture made with a sterile needle in the scale fragments. The sterile water was used as the negative control. The onion scales were incubated at room temperature. Strains that caused scale necrosis were considered pathogenic. The inoculated bacteria were re-isolated and confirmed from the symptomatic inoculated onion scales as described above. Four replicates per strain were used for one experiment, and the experiment was conducted twice.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eDifferences between means were assessed using the Newman-Keuls test at a significance level of 5%. Statistical calculations were performed for the experimental results presented in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding declaration\u003c/h2\u003e\n\u003cp\u003eThe studies were performed in the frame of project BioSafeFood \u0026ldquo;Development of a technology for producing high-quality, consumer-safe fruit and vegetables using new biopreparations to protect crops against diseases\u0026rdquo;, co-financed by UE from the European Regional Development Fund under the Smart Growth Operational Programme 2014\u0026ndash;2020, contract no. POIR.04.01.02-00-0100/17\u0026thinsp;\u0026minus;\u0026thinsp;00 NCBiR. (National Centre for Research and Development).\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eA.M. designed the project, analyzed the data, performed experimental studies and wrote the manuscript. J.P. designed the project, analyzed the data, performed experimental studies and wrote the manuscript. H.G. analyzed the data, performed experimental studies and wrote the manuscript. M.K. performed the experimental part devoted to the identification of bacteria, wrote the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe studies were performed in the frame of project BioSafeFood \u0026ldquo;Development of a technology for producing high-quality, consumer-safe fruit and vegetables using new biopreparations to protect crops against diseases\u0026rdquo;, co-financed by UE from the European Regional Development Fund under the Smart Growth Operational Programme 2014-2020, contract no. POIR.04.01.02-00-0100/17-00 NCBiR. (National Centre for Research and Development).All authors are the inventors of Polish patent entitled \u0026lsquo;Strains of the genus Pantoea spp. and the use of strains of the genus Pantoea spp. in plant protection\u0026rsquo; pat. no. 245234.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eIdentity based on 16S rDNA - GenBank accession numbersStrain 22ZK Serratia inhibens accession number PP660574Strain 24PK Serratia inhibens accession number PP660575Strain 131M Pseudomonas laurylsulfativorans accession number PP664062Strain 132M Serratia inhibens accession number PP660576Strain 133M Pseudomonas alvandae accession number PP664061Strain M13/07 Pantoea agglomerans accession number PP660862Strain T08/33 Pantoea allii accession number PP660869Strain T08/48 Pantoea allii accession number PP660868Strain T5/1 Pantoea ananatis accession number PP660863Strain T14/1A Pantoea ananatis accession number PP660867Strain T14/15 Pantoea allii accession number PP660866Strain T16/8 Pantoea pleuroti accession number PP660864Strain T14/8 Pantoea ananatis accession number PP660865\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAit Tayeb, L. 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A novel biocontrol strain \u003cem\u003ePantoea jilinensis\u003c/em\u003e D25 for effective biocontrol of tomato gray mold (causative agent \u003cem\u003eBotrytis cinerea\u003c/em\u003e). \u003cem\u003eBiol. Control\u003c/em\u003e. \u003cb\u003e164\u003c/b\u003e, 104766. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biocontrol.2021.104766\u003c/span\u003e\u003cspan address=\"10.1016/j.biocontrol.2021.104766\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"gray mold, antagonistic bacteria, biocontrol, Pantoea allii, Pantoea agglomerans, Pantoea ananatis","lastPublishedDoi":"10.21203/rs.3.rs-7696222/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7696222/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to isolate and evaluate bacterial strains with the potential to control storage diseases of apple trees. A total of 2,027 bacterial isolates were obtained from the phyllosphere of apple, strawberry, raspberry, and grape plants, as well as from soil associated with apple, strawberry, and raspberry crops. Screening using a dual-plate confrontation assay identified 1,678 isolates with antagonistic activity against \u003cem\u003eBotrytis cinerea\u003c/em\u003e, the causative agent of grey mold. Subsequent testing on apple fruit enabled the selection of 13 isolates exhibiting the highest biocontrol efficacy. These isolates were identified as belonging to the species \u003cem\u003eSerratia inhibens\u003c/em\u003e (22ZK, 24PK, 132M), \u003cem\u003ePseudomonas laurylsulfativorans\u003c/em\u003e (131M), \u003cem\u003ePseudomonas alvandae\u003c/em\u003e (133M), \u003cem\u003ePantoea agglomerans\u003c/em\u003e (T16/8, M13/07), \u003cem\u003ePantoea allii\u003c/em\u003e (T14/15, T08/33, T08/48), and \u003cem\u003ePantoea ananatis\u003c/em\u003e (T5/1, T14/1A, T14/8). The five most effective isolates (T5/1, T14/15, T16/8, T14/1A, and M13/07) reduced disease severity by 62.4% to 78.6%.\u003c/p\u003e\u003cp\u003eFurther antagonism assays against revealed that the selected isolates, besides \u003cem\u003eB. cinerea\u003c/em\u003e also strongly inhibited growth of \u003cem\u003eMonilinia fructigena\u003c/em\u003e and \u003cem\u003eColletotrichum acutatum\u003c/em\u003e in vitro.\u003c/p\u003e\u003cp\u003ePathogenicity tests confirmed that none of the 13 isolates induced hypersensitive reactions on tobacco or caused rot in potato tissues. However, five isolates (T08/33, T08/48, T5/1, T14/8, and T14/1A) were pathogenic in the red onion scale assay.\u003c/p\u003e","manuscriptTitle":"Screening and identification of epiphytic and soil bacteria beneficial in the control of postharvest apple disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 05:24:14","doi":"10.21203/rs.3.rs-7696222/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"83d1d743-835b-4b39-bb30-9ed2ed9bc7a6","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57410833,"name":"Biological sciences/Microbiology"},{"id":57410834,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-01-28T10:04:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 05:24:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7696222","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7696222","identity":"rs-7696222","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}