Identification of Antibiotics for Use in Selection of the Chytrid Fungi Batrachochytrium

1 Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium

2 dendrobatidis and Batrachochytrium salamandrivorans

5 Kristyn A. Robinson, Mallory Dunn, Shane P. Hussey, Lillian K. Fritz-Laylin*

8 The University of Massachusetts Amherst, Department of Biology, Amherst, MA 01003

9 *Correspondence: [email protected]

12 ABSTRACT

14 Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection

15 caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans

16 (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted

17 treatments are necessary to manage the disease. Currently, no tools for genetic manipulation

18 are available to identify and test specific drug targets in these fungi. To facilitate the

19 development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics

20 with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin.

21 We have identified effective concentrations of each for selection in both liquid culture and on

22 solid media. These concentrations are within the range of concentrations used for selecting

23 genetically modified cells from a variety of other eukaryotic species.

24 INTRODUCTION

26 Chytrids are early diverging fungi that are commonly found in aquatic and moist environments.1

27 They play key ecological roles, particularly by cycling carbon between trophic levels.2,3 Chytrids

28 have a biphasic life cycle characterized by motile and sessile stages (Fig. 1).4–6 They begin their

29 life as motile “zoospores,” which use a flagellum to swim through water and, for some species,

30 actin-based motility to crawl along surfaces.7,8 Zoospores then transition to a sessile growth

31 stage by retracting their flagellum and building a cell wall in a process referred to as encystation.

32 Encysted spores, known as sporangia, develop hyphal-like structures called rhizoids and grow

33 rapidly. Each sporangium produces many zoospores that exit via discharge papillae to begin the

34 life cycle anew.

36 Many chytrids are pathogens that infect protists, plants, algae, fungi, and vertebrates.2 The most

37 infamous chytrids are the vertebrate pathogens Batrachochytrium dendrobatidis (Bd) and B.

38 salamandrivorans (Bsal). Both pathogens cause chytridiomycosis, a skin disease plaguing

39 amphibians worldwide.4,6 Recent estimates indicate that Bd has affected several hundred

40 amphibian species and has been recorded on every continent except for Antarctica.9–11 Bsal

41 was more recently discovered in 2013 after a steep decline in fire salamander populations in

42 Belgium.6

44 Management strategies for these pathogens have been developed and implemented in limited

45 contexts, but implementation in real world settings remains a challenge. To develop better

46 treatments, we need to understand the biology of chytrids in order to identify targets for drug

47 development. However, studying the molecular mechanisms driving pathogenesis remains

48 challenging due to the lack of genetic tools available for chytrid fungi. The recent success in

49 genetic manipulation of a related chytrid species, Spizellomyces punctatus (Sp), is a major

50 breakthrough for our ability to study chytrid biology.8 We and others are now striving to adapt

51 this technology to Bd and Bsal to further our understanding of chytridiomycosis.

53 A key step to genetic tool development is the identification of methods for selection of

54 successful transformants. The most commonly used selection method is antibiotic resistance:

55 incorporating a gene that provides specific drug resistance allows transformed cells to survive

56 exposure to the antibiotic while all of the other cells are killed.12 Distinct classes of antibiotics

57 are commonly used for selection, each with their own molecular targets and corresponding

58 organismal specificity. In addition to testing whether a given antibiotic kills cells of interest, it is

59 important to pay attention to the effective concentration of each antibiotic. This is because a low

60 concentration will not apply sufficient selective pressure and a high concentration could produce

61 off-target effects and kill cells indiscriminately.13

63 In this paper, we examine five antibiotics used in fungal and animal systems and identify the

64 effective inhibitory concentration(s) necessary to prevent cell growth in liquid and solid media.

65 Hygromycin, Blasticidin, and Puromycin inhibit protein translation in both bacterial and

66 eukaryotic cells. Hygromycin inhibits protein synthesis by binding to the small ribosomal subunit

67 and stabilizing the tRNA in the A site, preventing the progression of translation.14 Blasticidin

68 inhibits the terminating step of translation while Puromycin causes the ribosome to prematurely

69 detach from mRNA.15,16 Although neomycin targets the prokaryotic 30S ribosomal subunit and

70 causes codon misreading and mistranslation, it has been used in eukaryotes because of the

71 similarity between mitochondrial and chloroplast ribosomes and bacterial ribosomes.17 Zeocin

72 intercalates in the DNA of both bacteria and eukaryotes and introduces double-stranded breaks,

73 ultimately causing cell death.18

75 RESULTS

77 To establish appropriate selection compounds for use with Bd and Bsal, we first identified

78 antibiotics commonly used for selection with both mammalian and fungal systems. We chose

79 five compounds (Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin) to test based on

80 the mechanism of action of each compound, their proven efficacy for use with both animal and

81 fungal cells, and the availability of resistance genes (Table 1). We next tested the ability of

82 these five compounds to inhibit the growth of Bd and Bsal cells in liquid culture. Although solid

83 agar media is typically used for colony selection in chytrid and other fungi,8,19,20 we chose to use

84 liquid culture to identify initial working concentrations because measuring zoospore release in

85 liquid media is rapid and easily quantified.

87 To measure the effect of each antibiotic on Bd and Bsal growth, we added a wide range of

88 antibiotic concentrations to cultures of age matched zoospores and allowed them to grow for

89 one full life cycle: three (Bd) or four (Bsal) days. We then measured the concentration of

90 released zoospores in each culture. Initial concentrations were selected based on known

91 inhibitory concentrations for other organisms (Table 1) and spanned many orders of magntude.

92 Based on these preliminary experiments (not shown), we then identified possible working

93 concentration ranges for each antibiotic in both species and tested intermediate concentrations

94 using three biological replicates separated in time (Fig. 2 and 3). To enable comparison of

95 zoospore release from replicate experiments conducted on different days, we normalized counts

96 for each replicate to its antibiotic-free control.

98 We identified antibiotic concentrations that consistently prevented growth in all three biological

99 replicates - the successful concentrations are highlighted in orange in each figure. We found

100 Hygromycin, Zeocin, Blasticidin and Neomycin could inhibit Bd growth in liquid culture (Fig. 2),

101 while all of the tested antibiotics inhibited Bsal growth (Fig. 3). In Bd, Hygromycin has the lowest

102 minimum inhibitory concentration (0.1 µg/ml), followed by Blasticidin (1 µg/ml), Zeocin (5 µg/ml),

103 and Neomycin (600 µg/ml). Puromycin did not inhibit growth in Bd with the concentrations

104 tested. In Bsal, Zeocin prevented growth at 1 µg/ml, followed by Blasticidin (2 µg/ml),

105 Hygromycin (10 µg/ml), Puromycin (50 µg/ml), and Neomycin (250 µg/ml).

106

107 Having identified working concentrations of these compounds for use with liquid media, we next

108 tested their efficacy on solid media. Growing cells on solid media allows for colony formation,

109 which is useful for isolating successful and independent genetic transformants by “picking”

110 colonies that grow under selection. To identify useful concentrations for selection on solid

111 media, we inoculated zoospores on nutrient agar plates containing varying antibiotic

112 concentrations. After a full growth cycle on selective media (three days for Bd, four days for

113 Bsal), we compared zoospore release to antibiotic-free control cultures by flooding plates with

114 water and looking for motile zoospores (Supplemental Videos 1 and 2). We defined successful

115 concentrations as those which yielded no zoospore release in either replicate. We found at least

116 one concentration for each antibiotic that prevented zoospore release in the timeframe of a

117 typical growth cycle (Fig. 4 and 5).

118

119 Because detection of colony formation often requires multiple growth cycles, we evaluated the

120 efficiency of growth inhibition by growing plates with no zoospore release for 14 days. We found

121 that all of the tested antibiotics inhibited Bd growth on solid media, but only Hygromycin,

122 Blasticidin and Zeocin inhibited growth in Bsal. For Bd, Hygromycin has the lowest minimum

123 concentration at 0.1 µg/ml, with Blasticidin and Zeocin both following at 10 µg/ml, Puromycin at

124 100 µg/ml, and Neomycin at 1 mg/ml (Fig. 4). In Bsal, Hygromycin, Blasticidin, and Zeocin all

125 prevented growth for at least 14 days at a concentration of 10 µg/ml, while Puromycin and

126 Neomycin did not prevent growth on solid media (Fig. 5). The recommended concentrations for

127 selection are highlighted in orange on the tables in both figures (Fig. 4B, 5B).

128

129

130 DISCUSSION

131

132 This study identified drug concentrations that reproducibly inhibited Bd and Bsal growth in either

133 liquid culture or on solid media. When a drug worked in both liquid culture and solid media, the

134 solid media typically required a higher concentration of antibiotic. This may be because of the

135 additional minerals found in the agar not present in the liquid media.21 Hygromycin, Zeocin, and

136 Blasticidin worked well for both species and at concentrations within the typical range used for

137 genetic selection in other species (Table 1). Puromycin and Neomycin were both able to inhibit

138 growth of Bd and Bsal, but required higher concentrations than are used for animal cell lines.

139 Although Hygromycin, Zeocin, and Blasticidin are all effective for preventing growth of Bd and

140 Bsal, we recommend first using Hygromycin for genetic selection because it has been

141 successfully used for selection of transformants in the nonpathogenic chytrid Spizellomyces

142 punctatus, and is widely used for other fungal species.8,22–24

143

144 The ability to select for genetically transformed cells will allow for tractable genetic models to

145 facilitate hypothesis testing in Bd and Bsal. The identification of useful selection agents and

146 appropriate working concentrations is an important first step in developing genetic tools for use

147 with Bd and Bsal. The natural step forward will be the design of selection cassettes, most

148 commonly in the form of transformation plasmids. We look forward to the development of these

149 and related molecular tools that will help us answer questions about the basic cell biology of

150 chytrids, fungal evolution, and amphibian pathology.

151

152

153 METHODS

154

155 Cell growth and synchronization

156 Batrachochytrium dendrobatidis (Bd) isolate JEL 423 was grown in 1% (w/v) tryptone (Apex Cat.

157 20-251) in tissue culture treated flasks (Cell Treat 229340) at 24 °C for three days. B.

158 salamandrivorans (Bsal) isolate AMFP 1 was grown in half-strength TGhL liquid media (0.8%

159 Tryptone, 0.2% gelatin hydrolysate, 0.1% lactose (w/v) in tissue culture treated flasks at 15 °C

160 for four days.25 For both species, we synchronized the release of motile zoospores by gently

161 washing the flask three times with fresh growth media and then incubating with 10 mL of media

162 for 2 hours. Age matched zoospores were then collected by centrifugation at 2000 rcf for 5

163 mins, resuspended in media, counted, and used for experiments as outlined below.

164

165 Drug treatments and quantitation for cells grown in liquid media

166 Neomycin (Fisher Cat. AAJ67011AE), Hygromycin B (Fisher Cat. AAJ60681MC), Blasticidin

167 (Fisher Cat. BP2647100), Puromycin (Fisher Cat. BP2956100), and Zeocin (Fisher Cat.

168 AAJ671408EQ), were screened for growth inhibition of Bd and Bsal. Cells were diluted to a

169 starting concentration of 5x105 cells/mL and 250 uL of cells were added to each well of a sterile

170 tissue culture treated 24-well plate (Cell Treat 229123). 250 µl of appropriately diluted antibiotics

171 were added to each well and mixed thoroughly. Plates were sealed with parafilm and grown at

172 either 24 °C for three days (Bd), or 15 °C for four days (Bsal). For each of three biological

173 replicates spaced in time, the concentration of released zoospores was estimated using a

174 hemocytometer. Zoospore concentrations were normalized to the no drug control and data

175 plotted using Prism (GraphPad v8).

176

177 Drug Treatments and Quantitation for cells grown on solid media

178 We added 1% agar to 50 mL batches of 1% tryptone (w/v) and half-strength TGhL then

179 autoclaved. Each antibiotic was added to a separate, pre-cooled, 50 mL batch of media, and 10

180 mL of the solution added to one of five 15 mm2 plates (VWR 25384-090) and allowed to solidify.

181 Plates were wrapped in parafilm and aluminum foil, and stored at 4 °C. Plates were inoculated

182 by evenly spreading 5.0 x 106 zoospores across the agar and incubated at 24 °C for three days

183 (Bd) or 15 °C for four days (Bsal). Zoospore release was evaluated by imaging each plate for 20

184 seconds at one second intervals using a Nikon Ti2-E inverted microscope equipped with 10x

185 PlanApo objective and sCMOS 4mp camera (PCO Panda) using white LED transmitted light.

186 Approximate zoospore activity was assessed as: 0 (no visible zoospores), + ( < 25% zoospore

187 activity of control plates lacking antibiotic), ++ (~50% zoospore activity of control plates), or +++

188 (equivalent zoospore activity to control plates). To determine the lowest antibiotic concentration

189 that could completely inhibit growth, plates that yielded “0” growth were allowed to grow for 14

190 days at the appropriate incubation temperature and reassessed as above.

191

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356

10 μm

Figure 1. Life cycle of chytrid fungi. As illustrated here with images of Bsal, chytrid fungi have a biphasic life cycle characterized by a stationary growth phase called a sporangium (top) and a motile dispersal phase called a zoospore (bottom). Images taken at 100X using differential inter- ference contrast (DIC) microscopy. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 2

AA Hygromycin B Zeocin 125 100

100 75 75 50 50 % Growth % Growth 25 25

0 0 0.00 0.05 0.10 0.15 05 10 0.0 0.5 1.0 1.5 5 10 Concentration (µg/ml) Concentration (µg/ml)

C Blasticidin D Puromycin 40 125

100 30 75 20 50 % Growth % Growth 10 25

0 0 0.00 0.25 0.50 0.75 1.00 1.25 5 6 0 50 100 150 200 250 Concentration (µg/ml) Concentration (µg/ml)

E Neomycin

150

100

% Growth 50

0 0 50 100 150 200 250 600 Concentration (µg/ml) bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 2. Inhibition of Bd growth in liquid media. Percent of Bd growth in liquid media supplemented with (A) Hygromycin, (B), Zeocin, (C) Blasticidin, (D) Puromycin, and (E) Neomycin as compared to an antibiotic free control for three temporally isolated replicates (circle, square, and triangle, shades of blue). Orange symbols indicate concentrations at which no growth occurred after three days in all three replicates. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 3

A Hygromycin B Zeocin 100 125

100 75 75 50 50 % Growth % Growth 25 25

0 0 0.0 0.5 1.0 8 10 12 0.00 0.01 0.02 0.5 1.0 10 Concentration (µg/ml) Concentration (µg/ml)

C Blasticidin D Puromycin 50 125

40 100

30 75

20 50 % Growth % Growth

10 25

0 0 0.0 0.1 0.2 2 0 5 10 50 100 250 Concentration (µg/ml) Concentration (µg/ml)

E Neomycin 150

100

% Growth 50

0 0 50 100 150 200 250 500 Concentration (µg/ml) bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 3. Inhibition of Bsal growth in liquid media. Percent of Bsal growth in liquid media supplemented with (A) Hygromycin, (B), Zeocin, (C) Blasticidin, (D) Puromycin, and (E) Neomycin as compared to an antibiotic free control for three temporally isolated replicates (circle, square, and triangle, shades of blue). Orange symbols indicate concentrations at which no growth occurred after four days in all three replicates. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 4

A +++ ++ }

+ 0

B Antibiotic Concentration Rep 1 Rep 2 (μg/ml) Hygromycin 0.01 ++ ++ 0.1 0 0 1 0 0 10 0 0 100 0 0 1000 0 0 Zeocin 0.1 + ++ 1 0 + 10 0 0 100 0 0 1000 0 0 Blasticidin 0.01 ++ ++ 0.1 + + 1 0 0 10 0 0 100 0 0 Puromycin 0.1 +++ +++ 1 +++ +++ 10 ++ +++ 100 0 0 1000 0 0 Neomycin 0.1 +++ +++ 1 +++ ++ 10 + +++ 100 + + 1000 0 0

bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 4. Inhibition of Bd growth on solid media. (A) Examples of Bd growth after three days on antibiotic selection plates. The ‘+’ demonstrates the relative zoospore activity of each plate compared to an antibiotic-free control plate. The box highlights zoospores, which appear as small dots while the bracket highlights sporangia. The zoospores in the ‘0’ image are immotile (see Supp Video 1). Scale bar 50 µm. (B) Bd growth on antibiotic selection plates. Concentrations highlighted in bold and orange are the lowest concentrations that prevent growth for at least 14 days post zoospore plating. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 5

A +++ ++

}

+ 0

B Antibiotic Concentration Rep 1 Rep 2 μg/ml Hygromycin 0.001 +++ +++ 0.01 +++ ++ 0.1 ++ + 1 0 0 10 0 0 Zeocin 0.01 ++ ++ 0.1 0 0 1 0 0 10 0 0 100 0 0 Blasticidin 0.01 + ++ 0.1 + 0 1 0 0 10 0 0 100 0 0 Puromycin 0.1 +++ +++ 1 ++ +++ 10 ++ + 100 + 0 500 0 0 Neomycin 0.1 ++ ++ 1 ++ ++ 10 ++ +++ 100 + + 1000 0 0

Figure 5. Inhibition of Bsal growth on solid media. (A) Examples of Bsal growth after four days on antibiotic selection plates. The ‘+’ demonstrates the relative zoospore activity of each plate compared to a no antibiotic control plate. The box highlights zoospores, which appear as small dots while the bracket highlights sporangia. The zoospores in the ‘0’ image are immotile (see Supp Video 1). Scale bar 50 µm. (B) Bsal growth on antibiotic selection plates. Concentrations highlighted in bold and orange are the lowest concentrations that prevent growth for at least 14 days post zoospore plating. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

357 TABLE AND SUPPLEMENTAL VIDEOS LEGENDS

358 Table 1. Antibiotic concentrations used to select for gene expression in select

359 eukaryotes. This table lists the key features of the antibiotics used in this study: the drug class,

360 the target, known resistance genes, the current listed price per gram from Millipore Sigma, and

361 the concentrations used in select eukaryotes. Species include representatives from plants

362 (Arabidopsis thaliana and Chlamydomonas reinhardtii), protozoa (Trypanosoma brucei),

363 amoebae (Dictyostelium discoideum), fungi (Aspergillus spp., Schizosaccharomyces pombe,

364 Saccharomyces cerevisiae), and animals (human) in addition to the two species tested in this

365 study. The lowest concentrations of each antibiotic which inhibited growth in liquid and solid

366 media for Bd and Bsal are listed from our findings in this study.

367 - no references were found.

368 * the organism had to be made susceptible for the antibiotic to work

369 ‡ the neo resistance gene is also used for resistance to the drug G418 which was not tested in

370 this study

371

Neomycin Hygromycin Blasticidin Puromycin Zeocin

Atypical Nucleoside Glycopeptide

Drug Class Aminoglycoside Aminoglycoside Antibiotic Aminonucleoside Antibiotic

Target Ribosome17 Ribosome14 Ribosome15 Ribosome16 DNA18

Known Resistance

Genes neo‡ hyg, hph bsr, bls, bsd pac ble

List price per gram $177/g

(MilliporeSigma) $1.93/g $998/g $6280/g $5340/g (Invivogen)

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.184721; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Lowest drug

concentration for Liquid: 600 Liquid: 5 Liquid: 10

growth inhibition µg/ml Solid: 1 Liquid: 1 µg/ml µg/ml Solid: Liquid: N/A Solid: µg/ml Solid:

for Bd mg/ml Solid: 0.1 µg/ml 10 µg/ml 100 µg/ml 10 µg/ml

Lowest drug

concentration for Liquid: 2 Liquid: 1

growth inhibition Liquid: 250 Liquid: 10 µg/ml µg/ml Solid: Liquid: 50 µg/ml µg/ml Solid:

for Bsal µg/ml Solid: N/A Solid: 10 µg/ml 10 µg/ml Solid: N/A 10 µg/ml

Concentration 100-200 10-20

used for HeLa cells - µg/ml26,27 µg/ml28,29 1-2 µg/ml30–32 50 µg/ml33

Concentration

used for Human

Embryonic stem

cells - 40 µg/ml34 2.0 µg/ml35 0.5-5 µg/ml35–37 300 µg/ml38

Concentration

used for

Fibroblasts - 40 µg/ml39 8 µg/ml39 2 µg/ml39 800 µg/ml39

Concentration

used for

Arabidopsis

thaliana - 15-50 µg/ml40,41 10 µg/ml42 - 100 µg/ml43

Concentration

used for

Dictyostelium

discoideum - 25-40 µg/ml44 10 µg/ml45,46 - 100 mg/L47

Concentration

used for

Trypanosoma 2-10

brucei - 5-50 µg/ml48,49 µg/ml50–52 0.1 µg/ml53 -

Concentration

used for

Chlamydomonas

reinhardtii 300 µg/ml54 1-20 µg/ml55 - - 5-15 µg/ml56,57

Concentration

used for 100 -125

Aspergillus spp 200-400 mg/ml58 100 µg/ml22 - - μg/ml59

Concentration

used for S. pombe 0.375 g/L60 400 mg/L23 30 µg/ml61,62 - 150 mg/ml63

Concentration

used for S.

cerevisiae 6.25 mM64 300 µg/ml24 10 mg/ml65 *200 uM66 -

372

373

374 Supplemental Video 1. Bsal zoospores with zero growth. Zoospores grown on antibiotic

375 selection plates are labeled “0” if no zoospores are released or zoospores showed no growth

376 and are immotile.

377

378 Supplemental Video 2. Bsal zoospores with “+++” growth. Zoospores grown on antibiotic

379 selection plates are labeled “+++” if the zoospore release is comparable to the no antibiotic

380 control.

381