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Potential control of potato soft rot disease by the 1
obligate predators Bdellovibrio and like organisms 2
3
Daniel Youdkes, Yael Helman, Saul Burdman, Ofra Matan, Edouard Jurkevitch 4
Department of Plant Pathology and Microbiology, Faculty of Agriculture, Food and 5
Environment, The Hebrew University of Jerusalem, Rehovot, 7610001, Israel. 6
7
8
Corresponding author: Edouard Jurkevitch 9
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Running title: Soft Rot Control 12
13
Keywords 14
Predators-prey-potato, Bdellovibrio, Rot bacteria, Predation resistance 15
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AEM Accepted Manuscript Posted Online 17 January 2020Appl. Environ. Microbiol. doi:10.1128/AEM.02543-19Copyright © 2020 American Society for Microbiology. All Rights Reserved.
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Abstract 18
Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. affect a 19
wide range of crops including potato, a major food crop. As of today, farmers mostly 20
rely on sanitary practices, water management and plant nutrition for control. 21
We tested the bacterial predators Bdellovibrio and like organisms (BALOs) to control 22
potato soft rot. BALOs are small, motile predatory bacteria found in terrestrial and 23
aquatic environments. They predate on a wide range of Gram-negative bacteria, 24
including animal and plant pathogens. To this end, BALO strains HD100, 109J and 25
merRNA were shown to efficiently prey on various rot-causing strains of 26
Pectobacterium and Dickeya solani. BALO control of maceration caused by a highly 27
virulent strain of P. carotovorum subsp. brasilense (Pcb), was then tested in situ 28
using a potato slice assay. All BALO strains were highly effective at reducing disease 29
up to complete prevention. Effectivity was concentration-dependent, and BALOs 30
applied before Pcb inoculation performed significantly better than when applied 31
after the disease-causing agent, maybe due to in situ consumption of glucose by the 32
prey, as glucose metabolism by live prey bacteria was shown to prevent predation. 33
Dead predators and the supernatant of BALO cultures did not significantly prevent 34
maceration, indicating that predation was the major mechanism for the prevention 35
of the disease. Finally, plastic resistance to predation was affected by prey and 36
predator population parameters, suggesting that population dynamics affects prey 37
response to predation. 38
39
Importance 40
Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. are 41
among the most important disease-causing bacteria in plants. Among other crops, 42
they inflict large scale damage to potatoes. As of today, farmers have little options to 43
control them. The bacteria Bdellovibrio and like organisms (BALOs) are obligate 44
predators of bacteria. We tested their potential to prey on Pectobacterium spp. and 45
Dickeya spp. and to protect potato. We show that different BALOs can prey on soft 46
rot-causing bacteria, and prevent their growth in-situ, precluding tissue maceration. 47
Dead predators and the supernatant of BALO cultures did not significantly prevent 48
maceration showing the effect is due to predation. Soft-rot control by the predators 49
was concentration-dependent, and higher when the predator was inoculated ahead 50
of the prey. As residual prey remained, we investigated what determines their level, 51
finding that initial prey and predator population parameters affects prey response to 52
predation. 53
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Introduction 57
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Potato (Solanum tuberosum) is a major food crop, the fifth economically important 59
crop with a global production of around 380 million tons in 2016 (FAO, FAO,2016. 60
http://www.fao.org/3/CA1796EN/ca1796en.pdf). It is estimated that up to 30% of 61
potato yields can be lost in the field and during storage (1). Main contributors to 62
spoilage are rot causing pectinolytic bacteria belonging to the Enterobacteriaceae 63
family like Pectobacterium spp. and Dickeya spp. (2). More specifically, these 64
pathogens induce bacterial black leg and soft rot diseases, which in addition to 65
potatoes, spoil a wide range of fruits, vegetables and ornamentals, including tomato, 66
onion, pepper, and cabbage (3), in a variety of climates. Pectobacterium 67
carotovorum has the widest host range of all the soft rot bacteria (3-5). The diseases 68
can be particularly destructive, causing total loses at any stage along the agricultural 69
production line (e.g. planting, during growing season, transport, and storage) (2). 70
They also have increased in prevalence in European countries and in Israel (6, 7) as 71
well as in previously unaffected areas (8). Climate change and global trade are 72
thought to enhance the conditions favorable for their proliferation and global 73
dispersal (9). 74
As of today there is no treatment that can stop disease progression once soft rot and 75
blackleg disease develop in a plant. Accordingly, control is largely reliant on 76
prevention, which is spread over various layers. For example, certification of seed 77
potatoes based on seed grade, which cannot detect latent infection of progeny 78
tubers from symptomless plants; dedication of crops for either seed to be used as 79
planting material for human consumption (10); and application of hygienic measures 80
such as washing and disinfecting machines used when planting, spraying, haulm 81
flailing, harvesting, and grading in store (4, 11). Also, good agricultural practices such 82
as well draining fields (4) and proper plant nutrition (12), positively affect the control 83
of rot diseases and reduce the risk of tuber decay. Several physical treatments to 84
control contamination of potato tubers by soft rot bacteria are practiced, including 85
hot water, steam dry hot air, UV and solar radiation. While the hot water treatment 86
is the most friendly from an environmental perspective, this method is unable to kill 87
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plant pathogenic bacteria that reside deep inside the tubers without negatively 88
affecting plant growth (10). At the other end of the production chain, good storage 89
procedures are an important factor in reducing disease (10). 90
Biological control offers an alternative to both physical and chemical controls, and 91
may enable disease prevention in contaminated seeds. It can rest on various 92
strategies, including antagonists which directly affect pathogen populations, 93
antibiosis, competition for nutrients, production of secondary metabolites, 94
interference with quorum sensing, and/or the induction of plant systematic 95
resistance (13-15). Bdellovibrio and like organisms (BALO) are a group of small, highly 96
motile Gram-negative bacteria, that predate exclusively upon other Gram-negative 97
bacteria and are found to be ubiquitous in many terrestrial and aquatic 98
environments (16). Most BALOs belong to the Oligoflexia (17) and like Bdellovibrio 99
bacteriovorus, the most studied of the BALOs, are periplasmic predators. This life 100
style is characterized by a typically small, highly motile cell, the so-called attack 101
phase, searching for a prey and, upon finding it, by penetration of and establishment 102
into the prey's periplasm. The predator then engages in filamentous growth at the 103
expense of the prey's content to finally divide, producing motile progeny (5 to 7 104
progeny cells with an Escherichia coli-sized prey, in 3 to 4h) that are released from 105
the dead prey cell (18). 106
BALOs exhibit strain-dependent prey range with susceptibility to a predator varying 107
between strains of the same species (19-21). Although the exploration of the 108
therapeutic potential of BALOs for medicine has dramatically increased in the past 109
years, showing that BALOs can curb infections in vivo (22-25), few studies have 110
explored their potential against phytopathogens (26-28). The im of this study was to 111
assess the efficiency of B. bacteriovorus against potato rot bacteria. We show that 112
various strains of the predators can prey upon the disease-causing agents. We 113
further show that B. bacteriovorus can completely prevent rot development in vivo. 114
The effect is concentration-dependent and is largest when the predator is applied 115
ahead of the pathogen. Finally, we tested a combination of B. bacteriovorus with the 116
gram positive Paenibacillus dentritiformis, a bacterium previously shown to reduce 117
soft rot disease in potato (29) . 118
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120
Materials and methods 121
122
Bacterial strains, growth media and growth conditions. The bacterial strains used in 123
this study are shown in Table 1. The prey bacteria P. carotovorum spp., Dickeya 124
solani and Pseudomonas corrugata, as well as the Gram-positive bacterium 125
Paenibacillus dendritiformis were grown in LB medium at 28oC in shaking flasks at 126
180 rpm. Escherichia coli was grown at 37oC. All were started from single colonies 127
originating from laboratory stocks kept at -80oC. Overnight cultures were centrifuged 128
at 4,200 g for 10 min at 4oC, resuspended in HEPES buffer amended with 2 mM CaCl2 129
and 3 mM MgCl2.7H20, pH7.4 (aHEPES). Optical density (OD) at 600 nm was adjusted 130
to OD600=10 by concentration, to provide a stock solution, which was stored at 4°C 131
until use, for up to two weeks. The bacterial predators Bdellovibrio bacteriovorus 132
HD100, HD100-Td-tomato, 109J, merRNA, and Micavibrio aeruginosavorus ARL-13 133
were from lab stock kept at -80°C. merRNA is putative standalone cyclic-di-GMP 134
riboswitch that is highly expressed during the attack phase (30); the merRNA 135
mutant exhibits increased motility (31). A bacteriological loop full from the frozen 136
stock of predators was transferred to a 250 mL Erlenmeyer flask containing 1 mL of 137
an OD600=10 prey suspension diluted in 25 mL of aHEPES. E. coli ML35 and P. 138
corrugata were used as prey for growth of B. bacteriovorus and M. aeruginosavorus, 139
respectively. The flask was shaken at 250 rpm and 28°C. Growth of the predator was 140
monitored by a decrease in turbidity of the suspension (due to lyses of the prey) and 141
visually verified by microscopy. After most of the prey cells were lysed, the 142
suspension was filtered through a 0.45 μm filter (Sartorius) to separate predatory 143
cells from any leftover prey. The filtrate was stored at 4°C until use for up to one day. 144
Prey and predator cultures were routinely counted by dilution plating as colony 145
forming units per ml (CFU.ml-1) and plaque forming units (PFU.ml-1), respectively. The 146
latter was performed in double layered agar (32). P. dendritiformis cultures were 147
counted using the most probable number (MPN) method. Bacterial suspensions 148
were ten times serially diluted in aHEPES, and 10 μL were dropped in a row (5 drops 149
per row) onto an LB agar plate and incubated at 28°C for up to two days. 150
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151
Heat-killing treatments and culture supernatants. When needed, prey and predator 152
cultures were killed by heating to 60oC for 30 and 50 min, respectively, in a Major 153
Science Elite dry bath block (USA). Supernatant from B. bacteriovorus cultures grown 154
as above were obtained by five times concentrating the culture and filtering thrice 155
through a 0.2 m filter (Sartorius, Germany). Aliquots were plated to confirm the 156
absence of propagules. 157
158
Predation dynamics in the presence of P. dentritiformis. B. bacteriovorus HD100-Td- 159
tomato, E. coli ML35, and P. dendritiformis were grown as above. The P. 160
dendritiformis culture was either concentrated or serially diluted to obtain various E. 161
coli:P. dendritiformis ratios upon mixing. Twenty µL of the predator, 100 µL of the 162
prey, 100 µL of P. dendritiformis and 780 µL amended HEPES (25 mM HEPES, 2 mM 163
CaCl2·2H2O, 3 mM MgCl2·6H2O, aHEPES) buffer were mixed. Growth of the predator 164
was tracked using fluorescence in a plate reader (33). Three experiments with three 165
replicates were conducted. 166
167
Construction of predators and prey strains expressing Td-tomato. One hundred µL 168
of a B. bacteriovorus 109J overnight culture was mixed with 200 µL of a ten-fold 169
concentrated E. coli SM10 donor culture carrying pMQ414::Td-tomato grown in LB 170
with 10 µg m-1 gentamycin (Gm) to an OD600= 0.4, and spread over a sterile 171
membrane (0.2 μm) placed upon a DNB (NB medium 10 times diluted) agar plate. 172
Following overnight incubation at 28 °C, the mixture was diluted in aHEPES and 173
plated in a double layer agar plates containing 10 µg.ml-1 Gn using E. coli SM10 as 174
prey. Lytic plaques indicating growth of the predator were screened for the presence 175
of the Td-tomato gene by PCR and for Td-tomato expression by fluorescence 176
microscopy (Ti-Eclipse, Nikon). 177
178
Sensitivity of the rot pathogens to predation. Five hundred microliters of OD600=10 179
prey culture, and 100 μL of a BALO lytic culture were diluted 50 times while mixed in 180
aHEPES buffer in 50 mL Falcon tubes, resulting in final concentrations of 181
approximately 108 CFU.ml-1 and 106 PFU.ml-1, respectively. The co-cultures were 182
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incubated at 28°C with constant shaking at 250 rpm, for up to 72 hours. Predation 183
was visually assessed by clarification of the suspension, and confirmed by phase 184
contrast microscopy, for the presence of a high density of predators (small, rapidly 185
swimming cells) and a low density of prey (larger cells). Further, predation by B. 186
bacteriovorus HD100, 109J and merRNA expressing Td-tomato- was tracked by 187
fluorescence (predator growth) and OD (prey decay) in a plate reader (Tecan Spark 188
10M, Switzerland) (33). Three experiments with three replicates were conducted for 189
each combination of predator and prey in both settings. 190
191
Potato slice assay. The red-skinned Desiree potato cultivar was sourced from local 192
supermarkets and used in all experiments. Potatoes were surface sterilized by 193
dipping one minute in a 1 % sodium hypochlorite solution, washed twice with sterile 194
distilled water and left to dry in a laminar flow hood. After potato surfaces were 195
dried, potatoes were washed with 70 % ethanol and again dried on sterile Petri 196
dishes. Slices of about 0.5 cm width were cut using a sterilized knife, and were 197
transferred using a sterile tweezer to Petri dishes containing sterile Whatman #1 70 198
mm diameter filter paper dampened with 330 μL of sterile distilled water. BALO 199
cultures were prepared as in the "bacterial strain" section, five times concentrated 200
to reach ~109 PFU mL-1, and diluted as required for inoculation at lower 201
concentrations. Pcb was grown as above, sub-cultured (~ 100 μL) in fresh LB to OD600 202
~ 0.2, washed twice in aHEPES buffer and the OD600 adjusted to 0.2, then diluted to 203
yield ~ 106 CFU mL-1. P. dendritiformis was grown overnight on an LB plate, 204
resuspended in aHEPES buffer to an OD600 adjusted to 0.2. Potato slices were 205
inoculated with 10 μL drops of the tested cultures applied to the center of the slice. 206
BALOs were either applied 60 min ahead of, or together with, or 60 min after Pcb. In 207
the appropriate experiments, P. dendritiformis was applied along with the predator. 208
The potato slices were incubated at 28°C for up to 48 h. All experiments were 209
repeated three times, with 10 replicates per treatment (unless otherwise specified). 210
After incubation, each slice was photographed with a Canon PowerShot S120 camera, 211
using a rule for scale. The maceration area was calculated using the ImageJ image 212
processing program. 213
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Bdellovibrio bacteriovorus survival on potato slices. Potato slices 0.5 cm thick and 215
1.7 cm wide were prepared using a sterile knife and a sterile cork borer. Bacterial 216
suspensions (109 PFU.ml-1) were inoculated into 0.5 cm thick slices for 0, 1, 24 and 48 217
h. The surviving predators were retrieved by vigorously vortexing the potato plugs 218
for ~7 min in 10 mL of HEPES buffer. Predators were quantified by plating, as 219
described above. 220
221
Predation persisters. The remaining Pcb population levels after predation by B. 222
bacteriovorus HD100 was measured by dilution plating. Predator and prey were 223
mixed at ratios varying from 1:1000 to 1000:1 and at concentrations of 106 to 109 224
PFU mL-1 predators and 105 to 108 CFU.mL-1 prey, denoted as B5 to B9, and P6 to P9, 225
respectively. 1:10 prey:predator ratios of varying inoculum concentrations were 226
incubated for 5 h; predator:prey ratios of 1:1, 1:10, 1:100, 1:1000 at predator 227
inoculum concentration of 106 PFU mL-1 were incubated for 24 h. 228
229
Effect of glucose on predator growth. The concentration of glucose was measured 230
at several sites in the potato slices using the InfinityTM Glucose Oxidase Liquid Stable 231
Reagent kit. aHEPES solutions including glucose at the measured concentrations 232
were prepared and used to measure the effect of glucose on predation, measured as 233
above. 234
235
pH measurements in potato slices. The pH was measured from the center, the edge, 236
and in blended potatoes, using MColorpHastTM non-bleeding pH-indicator strips 237
(Germany) with a pH range of 5 to 10 with 0.5 increments. The strips were compared 238
to measurements obtained with a Mettler Toledo SevenCompact S220 benchtop pH 239
meter (USA). 240
241
Statistical analysis. For the potato slice assay experiments, three or more repeated 242
experiments with ten replicates each were combined and analyzed in JMP 14 243
statistical software. Not all groups within the aggregates followed a normal 244
distribution and at least one group did not have equal variance, therefore 245
nonparametric comparisons were used, specifically the Steel-Dwaas all pairs test. 246
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Individual experiments were analyzed using the F-test and Student's t-test 247
(Supplementary material). Resistance levels were analyzed using the F-test or 248
Student's t-test. 249
250
Results 251
252
Soft rot disease bacteria are sensitive to BALO predation. Visual inspection of the 253
turbidity of cultures of the soft rot disease-causing bacteria into which a BALO was 254
inoculated showed that the tested predators preyed on all proposed prey, except M. 255
aeruginosavorus ARL-13, which was unable to consume D. solani (Table S1). 256
Predation dynamics were explored by tracking the growth of B. bacteriovorus strains 257
HD100, 109J and merRNA, expressing Td-tomato using fluorescence, and by the 258
concomitant decrease of the prey population by following turbidity (OD). B. 259
bacteriovorus HD100-Td-tomato grew as fast on Pcb and P. carotovorum subsp. 260
carotovorum WPP14 (Pcc WPP14) as on E. coli ML35 (a "standard" laboratory prey) 261
but exhibited a slight delay when preying on Pcc KC24 (Figure 1 A, B, D, E, F). B. 262
bacteriovorus HD100-Td-tomato predated much faster than B. bacteriovorus 109J- 263
Tomato on D. solani, reaching maximal population size within 40 h, while at 80 h 264
post-inoculation, maximal growth had not yet been reached by B. bacteriovorus 265
109J-Td-tomato (Figure 1 C, G). E. coli ML35 was preyed upon more efficiently by 266
both predators than D. solani (Figure 1 C, D). B. bacteriovorus merRNA-Td-tomato 267
was found to prey as effectively as B. bacteriovorus HD100-Td-tomato on Pcb (Figure 268
S1). The final yields of the predators, as measured by relative fluorescence units 269
(RFU) was above 108 cells.ml-1 with small variations (up 60% in cell number) (33). M. 270
aeruginosavorus ARL-13 predation on Pcb and Pcc was slow, taking about 40 h, and it 271
was unable to predate on D. solani, (Table S2). 272
273
P. carotovorum subsp. brasilense more virulent than D. solani in potato slice assays. 274
Pcb, a highly virulent pathogen (34) and D. solani, a disease-causing agent with 275
increasing spread (35), were compared for virulence in the potato slice assay. Pcb 276
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consistently induced more lesions than D. solani at each of the tested concentrations 277
(Table S3) and was selected for further work. 278
B. bacteriovorus protects potato slices from soft rot damage. Potato slice assays 279
were used to test the ability of B. bacteriovorus strains HD100, 109J and merRNA to 280
prevent maceration in situ. In all the experiments, the addition of 109 PFU.ml-1 of any 281
of the B. bacteriovorus strains at 60 min before inoculation with Pcb or dual 282
inoculation at the same time, significantly reduced (p < 0.02) the maceration area. 283
Yet, differences between strains were observed: HD100 and ∆merRNA were similarly 284
efficient at both inoculation times but 109J efficiency was reduced when applied 285
together with Pcb (Figure 2). When the predators where applied 60 min after Pcb, 286
∆merRNA was the only strain able to significantly reduce (p < 0.01) the macerated 287
area (Figure 2). 288
Next, different concentrations (106, 107, 108 and 109 PFU.mL-1) of B. bacteriovorus 289
HD100 were added 60 min before Pcb. The maceration area was significantly (p < 290
0.01) reduced at all concentrations as compared to the control (Pcb only-inoculated 291
treatment). The protective effect induced by the predator was concentration- 292
dependent, as the maceration area increased with decreasing predator 293
concentration (Figure 3). At 109 and 108 PFU.mL-1, maceration was completely 294
prevented, being significantly different (p < 0.01) from the unprotected control and 295
from the treatments with 107 and 106 PFU.mL-1 of the predator. However, even at 296
these lower B. bacteriovorus concentrations, a significant protective effect was 297
observed (Figure 3). The results presented here are averages of three independent 298
experiments (see Materials and Methods). Data from each individual experiment are 299
shown in the supplementary material (Figures S2 and S3). 300
Differential effects of live and dead predators, and of culture supernatant. Next, we 301
ascertained the role of predation in protecting the potato tissues against rot. 302
Explicitly, we aimed at verifying whether the observed effect was not the result of 303
indirect interactions such as elicitation of plant defenses. In that sense, the 304
supernatant of a B. bacteriovorus HD100 lytic culture, of heat-deactivated B. 305
bacteriovorus HD100 cells, and of live predator cells were inoculated onto potato 306
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slides 60 min before Pcb, i.e. according to the conditions that yielded the most 307
effective protection. As in the previous experiments, live B. bacteriovorus HD100 308
significantly reduced (p < 0.01) the maceration area compared to the Pcb-only 309
control. In contrast, the effect of the lytic culture supernatant and of the heat-killed 310
HD100 cells was not significantly different from that of the control with the 311
pathogen only (Figure 4), although the average maceration areas were consistently 312
lower in these treatments relative to Pcb-only controls in the three experiments 313
(Figure S4). 314
315
Adding P. dendritiformis to B. bacteriovorus does not improve protection. 316
Paenibacillus dendritiformis was previously shown to protect potato tubers against 317
soft rots (29). Since P. dendritiformis is Gram-positive and is therefore not a BALO 318
prey, we posited that combining both bacteria may augment protection. It was thus 319
important to ensure that neither bacteria has detrimental effects one on another. 320
The potential of P. dendritiformis to harm BALO prey strains through secretion of 321
metabolites was assessed by streaking LB plates previously inoculated with P. 322
dendritiformis with E. coli ML35, Pcb, Pcc WPP14 and Pcc KC24. Overall, no 323
detrimental effects were observed (Figure S5A). Interactions were further studied by 324
mixing P. dendritiformis and B. bacteriovorus HD100-Td-tomato at ratios of 1:1, 10:1 325
and 20:1, with E. coli ML35 as prey. Predation dynamics were slightly slower at the 326
10:1 and 20:1 ratios (Figure S5B). 327
328
Predator populations rapidly decrease in the absence of prey. In field settings the 329
timing of predator and prey interacting on a tuber or in the soil surrounding it is 330
impossible to control. We therefore measured the survival of B. bacteriovorus in situ. 331
The inoculum substantially decreased after 24 h by ~2 orders of magnitude to 107 332
PFU.ml-1, followed by another drop of 2.5 orders of magnitude in the following 24 h 333
while a control culture kept in aHEPES showed no such decrease (Figure S6). 334
335
Predation persisters are affected by predator:prey ratio. In BALO-prey interactions, 336
the prey population is not eradicated. The remaining population exhibits reversible, 337
plastic resistance (36), thus being a potential seed for regrowth of the pathogen onto 338
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the potato host. In order to understand if plastic resistance originates in a fixed ratio 339
of cells intrinsically less sensitive to predation or results from elicitation by predatory 340
activity, we examined the relationship between resistance, predator and prey 341
population sizes and their ratios. 342
At low concentrations of predator and prey, when the concentration of the former 343
was higher than that of the latter (B6:P5) or equal (B6:P6), the remaining prey 344
population size was similar to that of the inoculum or was slightly reduced. The 345
remaining prey level however, dropped by three and four orders of magnitude at 346
prey inocula 10 and 100 times higher, respectively, unrelated to predator 347
concentration (B7:P6 and B6:P7, and B8:P7 and at B6:P8, respectively). A further 348
increase in initial prey concentration reduced the ratio between initial and residual 349
population sizes, unrelated to initial predator inoculum levels (B9:P8 and B6:P9). 350
Finally at the high predator:prey ratio of B9:P6 residual prey were below detection 351
level (Figures 5, S7 and S8). 352
353
Glucose may inhibit predation in potato tissues. As the inoculation of B. 354
bacteriovorus after Pcb proved to be less efficient than the other treatments, and 355
since Dashiff et al. (37) had demonstrated that sugars can inhibit predation, we 356
tested the effect of glucose concentrations found in potatoes on predation. Glucose 357
concentrations from the center of the potato, the edge, and blended potatoes were 358
measured and found to be 3, 22, and 7 mM, respectively. pH measured on control 359
and macerated slices was consistently found to be 6 and 8, respectively (Figure S9). 360
Predation by B. bacteriovorus HD100 on live Pcb and E. coli ML35 at these glucose 361
concentrations was inhibited in a concentration-dependent manner. On heat- 362
deactivated E. coli ML35 cells, however, B. bacteriovorus HD100 grew as well as the 363
control, independent of glucose concentration (Figure S9). 364
365
Discussion 366
367
Being obligate predators of bacteria, BALOs have been dubbed "living antibiotics" 368
(38). This potential is being explored but mostly for applications in the medical field 369
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(22-25). Here, we show that BALOs are able to prevent bacterial infections in plant 370
tissues. The need for biocontrol approaches is particularly sore for bacterial diseases 371
of plants, as very few treatments exist, and most of the practices to keep crops 372
protected from such diseases, especially soft rots, are based on sanitation, nutrition 373
and growth management. 374
The different BALOs used on the potato slices were to different extents, able to prey 375
upon most of the pectinolytic bacterial strains tested. Predation was shown to stand 376
behind the protective effects against soft rot, as dead predators and a filtered 377
predatory culture supernatant (which also contained prey debris) were unable to 378
effectively protect the potato tissues against maceration However, this treatment 379
consistently reduced the average maceration area in the replicated experiments, 380
suggesting a mild protective effect may still be induced. Bacterial determinants such 381
as microbe-associated molecular patterns residues, including flagellum, EF-Tu or 382
exopolysaccharide may cause elicitation of plant immune response (39). Interestingly, 383
the flagellum of Bdellovibrio predators is sheathed (40). Moreover, their lipid A has a 384
unique composition, including mannose instead of phosphate residues (41). These 385
peculiar structural and chemical features may explain why in animals, exposition to 386
BALOs generate only minimal immune or inflammatory responses (23, 42, 43). We 387
propose that the low response observed may similarly be due to low levels of 388
defense elicitation in plants. 389
Differences were observed between predators, with M. aeruginosa showing a more 390
restricted prey range than B. bacteriovorus, and slower predation. It should be noted, 391
however, that BALO-prey interactions can be extremely specific since BALO strains, 392
even predators belonging to the same species, have differential affinities for strains 393
of prey belonging to the same species, varying from very "palatable" to highly 394
resistant to predation (20, 21). This was confirmed in this study as the different 395
BALOs, including closely related strains (B. bacteriovorus HD100 and 109J are >99% 396
identical at the genome sequence level (44)), showed differential efficiencies when 397
preying upon closely related strains of pathogens (e.g. Pcb, Pcc WPP14, KC24). 398
It should be noted that pathogen virulence did not affect BALO efficiency as the B. 399
bacteriovorus strains were more effective against the most virulent pathogen (Pcb) 400
in the potato slice assay than on the less virulent D. solani. Thus any particular BALO 401
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may not be a generic biocontrol agent, similarly effective against different soft rot 402
agents. While this may be viewed as a limitation in their use, and a cause of concern 403
for their application against D. solani, a pathogen with an increasing spread (35), it 404
should be pointed that novel predatory strains can be rapidly isolated against 405
emerging pathogens – as long as these are Gram-negative, providing a flexible and 406
"customized" answer to specific diseases. This approach is put in practice in the 407
development of BALO-based control of diseases in aquaculture (45, 46). 408
All three B. bacteriovorus strains (HD100, 109J, and the merRNA derivative of 409
HD100) were very effective in preventing maceration of potato slices when applied 410
before the pathogen or along with it, with 109J showing a somewhat reduced 411
performance in that latter setting. The effect was concentration-dependent, 412
completely preventing disease spread at high predator concentrations (109, 108 413
CFU.ml-1) and high predator:prey ratio (1000:1, 100:1, respectively) but also showing 414
significant efficiency at lower values (107, and 10:1; 106 CFU.ml-1 and 1:1). These 415
results are particularly remarkable as they further support predation as the main 416
mechanism standing behind the observed phenomenon. Indeed, at high predator 417
concentrations and high predator:prey ratio (tens per prey), prey cells may be 418
targeted by multiple predators at once, causing rapid premature lysis but preventing 419
predator multiplication. However, at low concentrations and ratios (1:1, 10:1), prey 420
lysis is not a concern (47). 421
An important feature in BALO-prey interactions is that prey populations are not 422
eradicated. Shemesh and Jurkevitch (36) discovered that the remaining prey cells in 423
a lytic culture were resistant to predation but that resistance was plastic and not 424
heritable. It was shown that it was not due to secreted compounds or residues found 425
in the lytic culture. Plastic resistance occurs in planktonic populations, as well as in 426
biofilm-forming prey populations (48, 49), and varies between predator and prey 427
combinations (20, 21). Consequently, resistant cells of the pathogen may remain, 428
constituting a potential for regrowth and infection. We further examined how 429
resistance is generated, showing that a low concentration of predator and prey 430
(B6:P5, B6:P6) was not conducive to predation. Low prey concentrations slow 431
predation (33, 50). This may explain that at B6:P5 and B6:P6, the original and the 432
final prey populations after incubation with the predator for 5 and 24 h were similar. 433
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Treatments in which the prey population was increased led to very large increases in 434
the ratio of initial to remaining prey population sizes that varied from about 103 435
(B7:P6, B6:P7) to above 104 (B8:P7,B6:P8). Since at these ratios and population sizes, 436
premature prey lysis is minimal (47), these findings suggest that the ratio of 437
resistance in the prey population is not a fixed proportion but a variable dependent 438
upon the number of predators and prey, and thus on population dynamics. The 439
absolute number of remaining prey cells also fluctuated but strikingly more so under 440
predator>prey ratio and short incubation time than under prey>predator ratios 441
(B6:P7, B6:P8) even though incubation was longer, further supporting that plastic 442
resistance results from the interaction of the predator with the prey. At the higher 443
ratios of B6:P9, predation may have not be complete within the time of the 444
experiment, and as mentioned above at B9:P6 prey cells may experience rapid lysis 445
(47). 446
The application of B. bacteriovorus HD100 and 109J 1h after Pcb resulted in a non- 447
significant decrease of the maceration area by only circa 20%. When the predator 448
was applied alone for the same amount of time on a potato slice, it survived and 449
generated plaques, demonstrating that loss of viability was not the cause of the loss 450
in efficiency. Moreover, B. bacteriovorus merRNA remained partly efficient under 451
the same conditions. However, longer exposure of the predator to starvation 452
conditions may have detrimental effects for field applications, an issue that should 453
be further evaluated. Taken together, these results suggest that the pathogen alters 454
the micro-environment, leading to the inhibition of predation. The growth of soft rot 455
bacteria on potato is accompanied by a shift from pH 6 to pH 8 in the tissues 456
resulting from the activity of quorum-sensing (QS) dependent, maceration-causing 457
lytic enzymes (4). The small inoculum used in our experiment (10 l of 106 CFU.ml-1, 458
i.e. 104 cells) spreads on the surface of the potato and may therefore not be 459
sufficiently dense to induce QS and thus to initiate pH increase in the micro- 460
environment within that short time frame. Glucose is present in the tissues that may 461
be readily used by Pcb, providing energy and carbon required for population 462
expansion. Glucose metabolism by soft rot-causing bacteria produces acids (51), 463
reducing pH which is a condition detrimental to predatory activity (37). Accordingly, 464
we posit that during the first phase of pathogen establishment, the micro- 465
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environment turns acidic, preventing predation. Supporting this, and as found by 466
Dashiff et al. (37), we observed that predation on dead prey cultures in the presence 467
of glucose was not inhibited. 468
Finally, the addition of the Gram-positive P. dentritiformis neither induced protection 469
nor improved the performance of B. bacteriovorus, rather causing slight, 470
concentration-dependent delays in predation, possibly through a decoy effect (52). 471
This shows that the combination of a Gram-positive biocontrol agent with a BALO 472
predator incurs a price. Depending upon the efficiency of the added strain against 473
the deleterious agent, this strategy may or may not be beneficial. Further strategies 474
to improve the efficiency of the BALOs may be explored, including cocktails of 475
predatory strains and their packaging, e.g. by encapsulation as recently described for 476
the protection of the whiteleg shrimp Penaeus vannamei from the pathogen Vibrio 477
vulnificus (53). In conclusion, we showed that BALOs have the potential to prevent 478
rot from spreading in healthy tissue and that protective action is more effective than 479
corrective action. 480
481
Acknowledgment 482
This research was supported by The Israel Ministry of Agriculture and Rural 483
Development as part of the “Root of the Matter- the Root Zone Knowledge Center for 484
Leveraging Modern Agriculture” (Core Knowledge Center grant 391/15). 485
486
487
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Tables 488
Table 1: List of bacterial strains and plasmids used in this work. 489
Strain / Plasmid Use Reference Escherichia coli ML35 Prey (54) SM10 Prey, conjugal transfer (55) Pseudomonas corrugata Prey Gyora Kryztman, ARO Pectobacterium carotovorum subsp. brasiliense (Pcb) Prey (56) subsp. carotovorum KC24 Prey (5) subsp. carotovorum WPP14 Prey (5) Dickeya solani Prey (7) Paenibacillus dendritiformis T Biocontrol agent (29) Bdellovibrio bacteriovorus HD100 Predator (57) HD100-Td-tomato Predator (58) 109J Predator (19) 109J-Td-tomato Predator (58) ∆merRNA-Td-tomato Predator This lab Micavibrio aeruginosa ARL-13 Predator (59) Plasmids pMQ414 Constitutive for Td-
tomato, Gm resistant (58)
490
491
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Legends to figures 492
Figure 1. Growth of B. bacteriovorus 109J-Td-tomato and HD100-Td-tomato and decay of 493 Pectobacterium carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum 494 WPP14 and KC24 (Pcc W and Pcc K, respectively)(A, B, E, F), E. coli ML35 (A, B, D, E, F,), and 495 Dickeya solani (Ds) (C, G) prey populations. The predatory populations were tracked by 496 fluorescence (relative fluorescence units, (RFU)(A, B, C, D), the prey populations by optical 497 density (OD)(E, F, G). 498 499 Figure 2. The effect of B. bacteriovorus strains HD100 (A), 109J (B), and ∆merRNA (∆mer) (C), 500 on tissue maceration in potato slices, induced by the soft rot bacterium P. carotovorum 501 subsp. brasilense (Pcb). B. bacteriovorus (10 ml, 109 PFU.ml-1) inoculated 60 min before, 502 together with or 60 min after Pcb (10 ml, 106 CFU mL-1). Five photographs of replicates out of 503 10 per treatment are shown in (D). Macerated tissue appears as dark patches. The results 504 shown are after combination of three independent experiments (four for HD100) with 10 505 replicates per treatment in each experiment. Error bars represent the standard error of 30 506 replicates (40 replicates for HD100). Treatments not connected by the same letter are 507 significantly different (p < 0.01 for HD100 and ∆merRNA; p < 0.02 for 109J) according to 508 Steel-Dwaas all pairs test. The independent experiments are shown as supplementary 509 material Fig. S2. 510 511 Figure 3: The effect of B. bacteriovorus strains HD100 inoculated at concentrations 106 ,107, 512 108, and 109 PFU.ml-1 (10 ml) on tissue maceration in potato slices, induced by the soft rot 513 bacterium P. carotovorum subsp. brasiliense (Pcb) (10 ml, 106 CFU ml-1) inoculated 60 min 514 after the predator. Five photographs of replicates out of 10 per treatment are shown in (B). 515 Macerated tissue appears as dark patches. The results shown are after combination of three 516 independent experiments with 10 replicates per treatment each. Error bars represent the 517 standard error of 30 replicates. Treatments not connected by the same letter are 518 significantly different (p < 0.01) according to Steel-Dwaas all pairs test. Independent 519 experiment are shown in Figure S3. 520 521 Figure 4. Effect of inoculating B. bacteriovorus HD100 (10 ml, 109 PFU.ml-1), heat-deactivated 522 (killed) HD100, and the supernatant of HD100 60 min before P. carotovorum subsp. 523 brasiliense (Pcb) (10 ml, 106 CFU mL-1) on soft rot disease development in potato slices. The 524 results shown are after combination of three experiments with 10 replicates per treatment. 525 Error bars represent standard error of 30 replicates. Treatments not connected by the same 526 letter are significantly different (p < 0.01) according to Steel-Dwaas all pairs test. Data from 527 each independent experiment are shown in Figure S4. 528 529 Figure 5. Surviving P. carotovorum subsp. brasiliense (colony forming units.ml-1) after mixing 530 with B. bacteriovorus HD100 in aHEPES at initial concentrations of: A. 106, 107, 108 and 109 531 PFU.ml-1 predators and 105, 106, 107 and 108 CFU.ml-1 prey, 5 hours (T = 5 h) after mixing 532 predator and prey. Predator: prey ratio was kept at 10:1. Horizontal red bars are the 533 concentration of the predator at the end of the experiment. B. 106 PFU.ml-1, predators and 534 106, 107, 108 and 109 CFU.ml-1 prey, 24 hours (T = 24 h) after mixing. B denotes predator, P 535 denotes prey, the number denotes the exponent of the initial concentration. The results 536 shown are after combination of three (A) and two (B) independent experiments. Error bars 537 indicate standard error. Within each treatment (BiPj) bars not connected by the same letter 538 are significantly different (p < 0.01). The independent experiments are shown in Figures S7, 8. 539
540
541
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