Introduction of a putative biocontrol agent into a range ...sri.haifa.ac.il/images/vered_2.pdf · Phytoplasma, the causative agent of bois-noir disease of grapevines, are vectored

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eIntroduction of a putative biocontrol agent into a range of Phytoplasma- and

Liberibacter-susceptible crop plants

A putative biocontrol agent can penetrate a wide range of crop plants

Ofir Lidor,1 Orit Dror,2 Dor Hamershlak,2,3 Nofar Shoshana,2 Eduard Belausov,4 Tirtza

Zahavi,5 Netta Mozes-Daube,1 Vered Naor,6,7 Einat Zchori-Fein,1 Lilach Iasur-Kruh8 &

Ofir Bahar2, †

1Department of Entomology, Agricultural Research Organization, Newe Ya’ar

Research Center, Ramat Yishai Israel

2Department of Plant Pathology and Weed Research, Agricultural Research

Organization, Volcani Center, Rishon LeZion, Israel

3The Robert H. Smith Faculty of Agriculture, Food and Environment, the Hebrew

University of Jerusalem, Rehovot, Israel

4Microscopy Unit, Institute of Plant Sciences, Agricultural Research Organization,

Volcani Center, Rishon LeZion, Israel

5Extension Service, Ministry of Agriculture, Israel,

6Shamir Research Institute, Katzrin, Israel,

7Ohallo College, Katzrin, Israel,

8Department of Biotechnology Engineering, ORT Braude College of Engineering,

Karmiel, Israel.

† corresponding author: [email protected] Dr. Ofir Bahar Department of Plant Pathology and Weed Research A.R.O. Volcani Center HaMakkabbim Road 68, Rishon LeZion P.O.B 15159 7528809 ISRAEL Tel: 972-3-9683561

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eAbstract

BACKGROUND

Phytoplasma, the causative agent of bois-noir disease of grapevines, are vectored by

the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). A Dyella-like bacterium

(DLB) isolated from H. obsoletus inhibits the growth of Spiroplasma melliferum, a

cultivable relative of phytoplasma. Additional evidence suggests that DLB can reduce

the symptoms of yellows disease in grapevine plantlets. The present aim was to test

whether DLB could colonize a range of Phytoplasma- and Liberibacter-sensitive crop

plants, and thus assess its potential agricultural use.

RESULTS

Vitex agnus-castus – the preferred host plant of H. obsoletus was found to be a natural

host of DLB, which was successfully introduced into a range of crop plants belonging

to seven families. The most effective DLB application method was foliar spraying.

Microscopy observation revealed that DLB aggregated on the leaf surface and around

the stomata, suggesting this is its route of entry. DLB was also present in the vascular

tissues of plants, indicating that it moved systemically through the plant.

CONCLUSIONS

DLB is a potential biocontrol agent and its broad spectrum of host plants indicates the

possibility of its future use against a range of diseases caused by phloem-limited

bacteria.

Keywords: biocontrol, endophytes, Phytoplasma, Liberibacter


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e1 INTRODUCTION

Candidatus Phytoplasma (Class: Mollicutes, hereafter ‘phytoplasma’) are Gram-

positive, wall-less, non-culturable phytopathogenic bacteria. Phytoplasma

transmission between plants strictly depends on phloem-feeding hemipteran insects,

mostly leafhoppers (Cicadellidae), planthoppers (Cixiidae, Delphacidae, Derbidae, and

Flatidae) and psyllids (Psyllidae).1 These insect vectors introduce phytoplasma cells

directly into the plant’s phloem sieve elements, where the pathogen multiplies and

spreads throughout the plant.2

Phytoplasma infects several hundred plant species, including perennial and annual

crops.3 Common phytoplasma-induced symptoms include leaf yellowing and curling,

shoot proliferation (witch’s broom), flower discoloration (virescence), and malformation

(phyllody).3,4 These abnormalities inevitably impair both quality and quantity of yield.

The most common means of controlling phytoplasma diseases is application of

insecticides to manage vector populations.1,5 The effectiveness of this approach is

limited, especially in perennial crops, where one transmission event by a single vector

is sufficient to cause infection.2 The absence of an epiphytic stage, in which the

bacterium is vulnerable to surface applications of chemicals or biological substances,

and the lack of known crop varieties that are genetically resistant to phytoplasma

diseases, leave farmers with very few options to control phytoplasma disease.1,5 Thus,

there is an urgent need to develop novel management tools against phytoplasma and

other phloem-inhabiting phytopathogenic bacteria. One such tool could be biocontrol

based on the use of beneficial bacteria.

Nonpathogenic endophytic bacteria inhabit plant inner tissues for at least part of their

life cycle without causing any apparent disease.6-9 The association of nonpathogenic

endophytes with plants benefits the latter by inducing plant growth through production

of plant phytohormones,7,10 improved nutrient availability,11,12 accelerated seedling

emergence,13 etc. In addition, some bacterial endophytes may protect plants from biotic

stresses via direct antibiotic activity, competition, and immune-response priming.14,15


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eOne example of the last is Burkholderia phytofirmans, an endophyte of grapevine that

inhibits Botrytis cinerea mold, directly by antifungal activity and indirectly by priming

H2O2 production and salicylic acid (SA) and jasmonic acid (JA) related gene

expression.16

Nonpathogenic endophytic bacteria also reduce phytoplasma-induced symptoms. For

example, phytoplasma-infected Catharanthus roseus (periwinkle) plants showed fewer

symptoms when inoculated with the biocontrol agent Pseudomonas migulae, strain

8R6.17 The authors speculated that the reduction in symptoms was due to direct

damage to phytoplasma cells and to the 1-aminocyclopropane-1-carboxylate (ACC)

deaminase activity of 8R6, which led to reduced ethylene levels, thereby delaying

disease symptoms.17 Another example is the co-inoculation of daisy plants with

‘phytoplasma asteris’, the causal agent of chrysanthemum yellows phytoplasma (CYP)

and Pseudomonas putida as a protecting agent. Although the presence of P. putida

did not appear to reduce phytoplasma titer, it improved plant growth compared with

that of plants inoculated only with ‘phytoplasma asteris’.18 The mechanism in this case

was not clear, but it was tentatively attributed to bacterial production of indole acetic

acid (IAA), which would compensate for the phytoplasma-induced deficiency of the

phytohormone in infected plants.18

We have recently isolated a Gram-negative, Dyella-like bacterium (DLB) from

Hyalesthes obsoletus (Hemiptera: Cixiidae), the vector of bois-noir phytoplasma in

grapevines.19 This indicates that H. obsoletus can carry both DLB and bois-noir

phytoplasma; however, it is not known whether the two bacteria simultaneously co-

inhabit H. obsoletus. In Israel, H. obsoletus thrives and completes its life cycle on Vitex

agnus-castus (Abraham’s balm) shrubs, but it cannot complete its life cycle on

grapevines.20 Conversely, although this planthopper can transmit phytoplasma to

grapevine, phytoplasma were never detected in Abraham’s balm shrubs in Israel,20 but

they were detected in Abraham’s balm shrubs in Montenegro.21 Whether DLB is


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etransmitted to and/or acquired from Abraham’s balm plants by H. obsoletus is not

known.

In a recent study,22 we demonstrated that DLB can penetrate grapevine plantlets when

applied by either foliar spraying, soil drenching, root dipping, or injection. Microscopic

and PCR analyses of DLB-inoculated grapevines indicated that DLB cells were located

inside the phloem tissue and could persist for up to a month following application.22

Importantly, DLB inhibited the growth of the Mollicute Spiroplasma melliferum19 in vitro,

and markedly reduced phytoplasma-induced symptoms in grapevine plantlets.22

If DLB prove to be an efficient biocontrol agent, it would be essential to determine its

rate of establishment in relevant susceptible crop plants. In the present study, we

examined the colonizing ability of DLB in a range of crop plants and one environmental

host (Abraham’s balm). We also examined DLB host penetration pathways,

localization patterns and in planta persistence following various inoculation

procedures. Our results indicate that this newly-discovered bacterium – and possible

biocontrol agent – is a natural plant colonizer with a potentially very broad host range.

Furthermore, the complete genome of DLB was recently sequenced and deposited in

the Genbank (NCBI) under accession no. LFQR00000000.23

2 MATERIALS AND METHODS

2.1 Bacterial strains and inoculum preparation

DLB stocks were kept at -80C in a 30% glycerol stock and were freshly streaked on a

modified CCT-agar,19 composed of sucrose (67 g L-1), sorbitol (10 g L-1), Lysogeny

Broth (LB, Difco) (2 g L-1) and Bacto-agar (13 g L-1), and cultivated at 28˚C. Liquid DLB

cultures were prepared by inoculating a 3-5 mL starter containing Nutrient Broth (NB,

Difco) or LB media with a single DLB colony from agar plates. The starters were

cultivated for ~24 h at 28ºC with shaking (220 rpm) until the culture was turbid; they

were then used to inoculate the necessary volume of broth culture at a ratio of 1:100


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e(V:V) in an Erlenmeyer flask. The DLB cultures were cultivated until they reached a

concentration of ~108 colony forming units (CFU) mL-1 as determined by counts

following serial dilution and plating of cultures on CCT-modified plates incubated for

48 h at 28ºC.

2.2 Generating a gfp-expressing DLB strain

To monitor DLB localization in planta, a gfp-expressing strain (DLBGFP) was generated.

DLB cells were transformed according to Enderle et al (1998).24 In brief, DLB was

streaked on a CTT-modified agar plate and grown at 28ºC for 48 h. A 3-mm colony

was lifted with a sterile loop, and transferred into a 1.5-mL Eppendorf tube containing

500 µL of double-distilled water (DDW). Tube contents were centrifuged at 16,000 x g

(~12,300 rpm – Eppendorf centrifuge model 5810R, rotor F45-30-11) for 1 min and the

supernatant was discarded. The cell pellet was resuspended with 500 µL of DDW,

further washed with a repeated centrifugation step, and finally resuspended in 50 µL

of DDW. The cells were kept on ice and a plasmid harboring a green fluorescent protein

(gfp) and a kanamycin-resistance gene (‘pKT-Kn’)25 was added to the tube. The

mixture of cells and plasmid was then transferred to an electroporation cuvette and

placed in an electroporator (ECM 399 model, BTX, Harvard Apparatus, Holliston, MS,

USA). Electroporation was set at 150 Ω, 36 μFD, and field strength of 1.8-2.5 kV was

obtained, depending upon the gap size of the cuvette (1 mM gave 1.8 kV; 2 mM, 2.4

kV). A 1-mL aliquot of S.O.C medium was immediately added to the cuvette and then

collected and transferred into a 15-mL tube for incubation (1 h at 28˚C, 220 rpm).

Electroporated cells were then plated at two volumes (20 and 200 µL) on CCT-modified

agar plates containing kanamycin (50 µg mL-1). DLB colonies growing on the

kanamycin plates were confirmed to carry the ‘pKT-Kn’ plasmid by confocal

microscopy.


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e2.3 Abraham’s balm shrubs and H. obsoletus insect sampling

Abraham’s balm shrubs grown outdoors at the Newe-Ya’ar Research Center (originally

collected in 1996 near the Kishon River) were used to determine whether DLB naturally

colonizes Abraham’s balm shrubs. The plants were drip-irrigated continuously

throughout the year (10 m3 per 3 weeks) apart from the winter months (December-

April). Four Abraham’s balm shrubs were sampled monthly from September to

December (2016) and again in April through June (2017); they were not sampled

during January through March because Abraham’s balm is a deciduous shrub and was

leafless during those months. from each sampling date, two leaves were randomly

collected from each plant and DNA was extracted from each leaf separately (n = 8).

DLB presence was determined by PCR with specific primers (as described in 2.6).

To assess the prevalence of DLB in H. obsoletus feeding on DLB-infected Abraham’s

balm shrubs, insects were collected at the same site in Newe Ya’ar in October 2016,

with a vacuum unit comprising a modified Echo model PB 1000 leaf blower, in which

the air intake and exhaust ports were interchanged as described elsewhere.26

Captured insects were anesthetized (-20ºC, 30 min) and H. obsoletus individuals were

separated from other insects. Ten H. obsoletus adults were recovered and placed

directly in 96% ethanol pending assay for the presence of DLB (see 2.6).

2.4 Plant material and growth conditions

Seeds of tested plant species (Tables 1 to 3) were sown in three types of substrate:

commercial potting soil (light-medium clay containing 63% sand, 12% silt, and 22%

clay, Gal-Marketing Ltd, Israel); perlite supplemented with NPK (20:20:20) fertilizer and

0.5% Hoagland agar,30 as indicated in Tables 1 to 3. Cucumis melo (melon), Cucumis

sativus (cucumber), Gossypium (cotton), Capsicum annuum (pepper), Nicotiana

benthamiana, Catharanthus roseus (periwinkle), and Sesamum indicum (sesame)

plants were grown under controlled conditions at 25-28ºC under cool white neon


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efluorescent illumination (14/10 h light/dark cycle) at approximately 200 μmol m-2 s-1 light

intensity. Daucus carota (carrot), Solanum lycopersicum (tomato) and Solanum

tuberosum (potato) plants were grown under natural light (approximately 11/13 h

light/dark cycle) with controlled temperature (25-28 ºC). The plants were treated with

DLB (as described in 2.5) at the 4-5th true-leaf stage, and kept under the same growth

conditions. Citrus grandis (pomelo) and Citrus sinensis (orange) plants were grafted

on bitter orange and grown outdoors in 10-L buckets containing commercial potting

soil under drip-irrigation, and DLB was applied when they were 6 years old.

2.5 DLB application methods

Two DLB application methods, namely soil drenching and foliar spraying, were tested

to determine the preferred method to introduce DLB effectively into plant tissue. In both

methods, DLB cultures were prepared as described in section 2.1, and applied to

plants at a concentration of ~108 CFU mL-1. For foliar spraying, a surfactant (either

Tween-20 [Acros Organics, NJ, USA] at 0.1% or Silwet L-77 (Adama, Israel) at 0.07%)

was added before application; the foliage was sprayed with a 1-L hand sprayer until

runoff. For soil drenching 50 mL of DLB culture was poured directly into the potting

medium of each plant. Untreated plants were used as controls.

2.6 Total DNA extraction and DLB detection in plants and insects

To determine the presence of DLB in plant tissue, samples were collected at several

time points, as indicated in Tables 1 to 3. Leaves were surface-sterilized by immersion

in 70% ethanol for 30 s, followed by a 2-min wash in 0.6% NaOCl and two additional

10-s washes with DDW.27 Then, 300-400 mg of leaf tissue were used for total DNA

extraction by means of a cetyl trimethyl ammonium bromide (CTAB) protocol described

elsewhere.28 DNA extraction from pepper samples was conducted with a plant/fungi


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eDNA isolation kit (Norgen Biotek, Thorold, Canada), according to the manufacturer’s

guidelines.

DNA from H. obsoletus insects was extracted by grinding each insect in 120 µL of lysis

buffer29 (5 mM of Tris-HCl, pH 8.0, containing 0.5 mM of EDTA, 0.5% of Nonidet P-40,

and proteinase K at 1 mg mL-1). The lysate was then incubated at 65 ºC for 15 min,

followed by 95 ºC for 10 min and then briefly centrifuged at 10,000 x g to pellet the

debris. A 3-µL aliquot of the aqueous supernatant were used as the template for PCR

analysis in a final volume of 25 µL. All DNA-lysate samples were kept at -20 ºC pending

screening for DLB.

The presence of DLB was determined by PCR with species-specific DLB primers

(DLBF, 5’-CTCTGTGGGTGGCGAGTGGC-3’ and DLBR, 5’-

ACCGTCAGTTCCGCCGGG-3’), as described elsewhere.19 PCR products of

environmental samples (i.e. Abraham’s balm shrubs and H. obsoletus insects) were

further tested by DNA sequencing by using BigDye Terminator v3.1 Ready Reaction

Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions, in a 3130xl

Genetic Analyzer (Applied Biosystems).

2.7 Visualization of GFP-labelled DLB by confocal microscopy

In planta localization of DLB was examined in carrot and sesame plants by using a gfp-

expressing strain (DLBGFP) and confocal microscopy. For carrot, seeds were sown in

perlite medium and grown as described in section 2.4 until the 3-4 true-leaf stage and

then spray- or drench-inoculated with the DLBGFP strain. Transverse and longitudinal

sections of petioles and leaves were taken 48 h post inoculation.

For sesame, seeds were sown in Hoagland agar and grown under fluorescent

illumination as mentioned in section 2.4, until the emergence of two true leaves.

DLBGFP was applied to 15 plants by gently brushing a single leaf of each plant with a

cotton swab soaked in the bacterial suspension. Five plants were sampled at each


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etime point, as indicated in Table 3. All samples were analyzed with an IX 81 confocal

laser-scanning microscope (Olympus, Tokyo, Japan).

2.8 Visualization of DLB with a scanning electron microscope

A scanning electron microscope (SEM) was used to visualize DLB on the leaf surface.

Two DLB-spray-inoculated sesame plants and one untreated plant were used.

Specimens were collected 3, 7 and 10 days post inoculation and prepared by chemical

fixation of plant leaves in a fixative solution (formaldehyde:glacial acetic acid:70%

ethanol, 5:5:90) for 24 h followed by dehydration with increasing amounts of ethanol

according to published procedures.31 The specimens were then critical-point dried with

a K850 Critical Point Drier (Quorum Technologies Ltd, Laughton, England) and sputter-

coated with gold by using a SC7620 mini-sputter coater (Quorum). They were

visualized with a TM3000 table top scanning electron microscope (Hitachi-High

Technologies, Toronto, Canada).

2.9 Statistics

The Pearson chi-square test was applied to determine statistical significance (α = 0.05)

of differences between treatments (inoculation type and growth media), using JMP

software (SAS, Cary, NC, USA) (Tables 1 to 3).

3 RESULTS AND DISCUSSION

3.1 DLB was present in environmental samples of Abraham’s balm shrubs

DLB was first isolated from the insect vector H. obsoletus. To determine whether DLB

also colonizes plants, we tested environmental samples of Abraham’s balm, the

preferred host for H. obsoletus in Israel; we detected DLB in all four shrubs tested from

September through December 2016 (Supporting information Fig. S1A), in one shrub

in April 2017 and in all shrubs in May and June (Data not shown). Nine out of ten H.


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eobsoletus individuals collected from the same Abraham’s balm shrubs in October 2016

were positive for DLB (Supporting information Fig. S1B). DLB-positive PCR bands from

each of the sampling points on all four shrubs were sequenced and confirmed to be

DLB.

The fact that DLB could be found in environmental samples of Abraham’s balm shrubs

indicates that DLB is a natural endophyte. This result, in light of our previous finding of

DLB in H. obsoletus gut, might suggest that DLB can be acquired and transmitted by

feeding insects, but this was not empirically tested. An example of a phloem-feeding

insect vector transmitting endophyte populations from source to recipient plants has

recently been reported.32 Intriguingly, although both DLB and phytoplasma were found

in H. obsoletus, unlike DLB, phytoplasma could never be detected in Abraham’s balm

shrubs in Israel.20

The presence of DLB in Abraham’s balm plants from September through December

and from April through June may suggest that it is persistent in this host. The low

incidence of DLB in Abraham’s balm in April can perhaps be attributed to the fact that

Abraham’s balm is a deciduous plant, which remains leafless from January through

March. One possible explanation is that during this leafless period DLB cells hibernate

in Abraham’s balm stems and/or roots and then become active again when new leaves

emerge in spring. Therefore, in April, although leaves were already present, DLB may

not have propagated to high levels and their distribution in the plant would be limited;

hence, the low detection rate. Another possibility is that DLB was lost from Abraham’s

balm shrubs during winter and was re-inoculated onto the shrubs with the reoccurrence

of H. obsoletus in spring. According to this scenario, there would be relatively little time

from March to April for the DLB population to build up, hence the low detection rate.

3.2 DLB penetration to various host plants


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eThe fact that DLB is a natural plant colonizer, together with its putative biocontrol

applications, has encouraged us to examine additional potential plant hosts to which

DLB could be applied. Twelve different plant species were inoculated with DLB by foliar

spraying or soil drenching, and then examined by PCR for the presence of DLB, which

was detected in the majority (9/12) of the tested plant species, as elaborated in Tables

1 to 3. DLB could not be detected in tomato, potato, or pepper, all belonging to the

Solanaceae family; but it was detected in N. benthamiana, which belongs to the same

family. These results suggest that when applied exogenously, DLB can colonize

multiple plant species, including annual and perennial crops. Our detection of DLB in

plant tissues following a fairly aggressive leaf-washing procedure suggests that DLB

was present, not merely on the leaf surface, but that it penetrated and established itself

inside the plant tissue. Furthermore, the fact that DLB could be detected in the foliage

of some plants following soil drenching supports previous observations19 that it also

penetrated the root system and systemically moved throughout the plant.

Whereas some endophytes, such as Azoarcus sp.,33 have a relatively restricted host

range and can colonize only grasses, DLB appears capable of adapting to many host

environments, at least for short periods of time. Although this trait is not unique to DLB

and is shared with other broad-host-range endophytes, such as B. phytofirmans,15 it is

an important trait to consider if DLB were to be used as a biocontrol agent.

3.3 DLB application and persistence in plants

To better understand the dynamics of DLB colonization in plants and to determine the

optimal application method, we conducted further experiments with two plants species:

carrot (Daucus carota) and sesame (Sesamum indicum) (Tables 2 and 3, respectively).

These plants were selected for further analysis specifically because both are known to

suffer from phloem-restricted pathogens such as phytoplasma4 and Ca. Liberibacter


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esolanacearum.34 The experiments described below wills facilitate future examination

the ability of DLB to suppress disease in these two crops.

The inoculation method used significantly affected in planta detection of DLB in both

plant species. In carrot experiment I, the DLB detection rate was similar for both foliar

spraying and drenching inoculations; in experiment II, however, foliar spraying resulted

in a significantly higher DLB detection rate than drenching, at all four time points (Table

2). Soil-grown sesame plants also had higher DLB detection rates following foliar

spraying at three of four sampling time points, but for those in Hoagland medium the

results of the two respective inoculation methods did not differ (Table 3). These results

suggest that under non-sterile conditions, DLB establishes more effectively in the plant

when applied by spraying than by drenching. The soil drenching findings revealed that

DLB can penetrate through plant roots and move systemically through the vascular

tissue of the plant.

The decline of DLB populations observed in both carrot and sesame was also

observed in grapevines, where DLB was not detected 37 days post inoculation.22 It is

possible that despite the ability of DLB to colonize various plant species, many of these

species cannot support its reproduction and survival for long periods. Additionally, non-

natural hosts of DLB may activate their defense systems against DLB, and thereby

lead to its decline in the plant, as was observed in other transient endophytes.35 DLB

appears to adapt more successfully to Abraham’s balm, where it was detected during

several months; this supports the notion that Abraham’s balm is a natural host of the

bacterium. In light of possible use of DLB as a biocontrol agent these findings imply

that DLB should most likely be reapplied at certain time intervals to maintain an

effective titer in the plant.

3.4 Microscope analyses of DLB localization in planta


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eThe results presented above indicate that DLB can penetrate the leaf surface and

move systemically within a plant. To better understand its penetration routes and

localization, a gfp-expressing DLB strain was applied to plants by either foliar spraying

or soil drenching, and was followed by both confocal microscopy and scanning electron

microscopy (SEM).

Time-scale monitoring of DLB following its application to sesame leaves showed that

DLB cells were randomly spread on the surface of the leaf by 3 days post inoculation

(Fig. 1); by seven and ten days post application, they were located mainly in the leaf

stomatal opening and partly in the intercellular spaces of the leaf cortex. SEM

observations agreed with confocal microscopy results in showing that DLB was located

around the stomatal openings of sesame leaves 3 days post exposure (Fig. 2A). Seven

and ten days post exposure DLB was clearly seen inside the stomatal openings (Fig.

2B, 2C). Bacterial cells were not observed in the control plants (Fig. 2D).

DLB was also detected in sesame roots, mainly in the outer periderm and in the inner

cortex layers, 10 days post exposure (Supporting information Fig. S2). Visual

examination of the root external surface (Supporting information Fig. S2A, washed

once in DDW), revealed that DLB was located around the root epidermal surface,

including the root hairs. Sections of the root reveal DLB also in the inner-cortex

(Supporting information Fig. S2B).

In carrots, DLBGFP applied to foliage by spraying could be easily detected covering the

leaf surface of unwashed leaves 48 h post inoculation (Fig. 3A, B). Leaf cross-sections

revealed that DLBGFP penetrated the leaf tissue within 48 h of inoculation and was

located in the xylem vessels and phloem cells (Fig. 3C, D). DLBGFP was also seen in

the xylem vessels of washed petioles (Fig. 3F). This further supports the notion of

systemic movement of DLB. The bacterium could not be observed in three attempts

(on three different plants) following soil drenching. In general, DLBGFP was unevenly


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espread in carrot tissue and could not be detected by microscopic analysis of numerous

PCR-positive samples.

Both nonpathogenic endophytes and phytopathogenic bacteria gain entrance into

plant tissue via natural openings such as stomata and through wounds.36 Our

microscopic observations support a similar penetration route for DLB; it could be seen

aggregating in stomatal openings. Furthermore, localization of DLB inside the vascular

tissue accounts for the systemic movement of DLB within plants, and is consistent with

the behavior of other endophytes, which migrate within the plant tissue either

apoplastically or through the vascular tubes.37 In light of the fact that H. obsoletus feeds

on phloem tissue, one could expect that this would be the niche colonized by DLB in

environmental samples of its Abraham’s balm host; but this has yet to be

demonstrated. Conversely, the localization of DLB in the phloem tissue following foliar

spraying is very intriguing. Very few bacterial genera are known to inhabit intracellular

phloem; they include obligate bacterial parasites such as phytoplasma and

Liberibacter, both non-culturable organisms that possess reduced genomes and that

are directly introduced into the phloem tissue by phloem-feeding insects. However,

DLB appeared to make its own way into the phloem tissue, even without the aid of a

phloem-feeding vector. If indeed DLB could be targeted to the phloem tissue, it would

represent a very elegant method to combat phloem-limited bacterial pathogens

specifically, with minimal side effects.

4 CONCLUSIONS

The present study has added an important element to the prospective use of DLB as

a biocontrol agent: DLB has a broad range of host plants, and it localizes to the same

plant tissue as its target pathogen. Thus far, the effectiveness of DLB as a biocontrol

agent was shown in vitro against S. melliferum and recently with phytoplasma-infected


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egrape plantlets. The present study widens the prospect for further testing the potential

use of DLB as a biocontrol agent in to protect numerous plant species.

ACKNOWLEDGEMENTS

The authors would like to thank Shira Gal for technical assistance with SEM operation,

Dr. Eric Palevsky and Sharon Hecht for supplying citrus plants, Dr. Phyllis Weintraub

for insect descriptions and scientific advice, Tamar Zakai for linguistic editing, and Dr.

Zvi Peleg for supplying sesame seeds. This work was supported by the Chief Scientist

of Israel’s Ministry of Agriculture and Rural Development and by Israel’s Ministry of

Economy and Industry.

This paper is contribution number 574/17 from the Agricultural Research Organization,

Volcani Center, Rishon LeZion, Israel.

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Entomol 51:91–111 (2006).

2. Christensen NM, Nicolaisen M, Hansen M and Schulz A, Distribution of

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eFigure legends

Figure 1: Localization of DLBGFP in sesame leaves over time. Hoagland-grown

sesame seedlings were inoculated by gentle brushing of the first two true leaves with

a DLBGFP soaked cotton swab. Upper panel (A) represents the upper epidermis layer

and lower panel (B) represents the mesophyll cells imaged at 3, 7, and 10 days post

inoculation. Neg indicates untreated control leaves (St indicates stomata). Images

are representative of five plant repetitions. Red fluorescence results from auto

fluorescence of chloroplasts and green fluorescence from DLBGFP cells. All images

are merges of red auto-fluorescence, GFP fluorescence and bright-field. All images

were taken via confocal microscope.

Figure 2: SEM pictures of DLB on sesame leaf surface. Leaves were sampled at 3

(A), 7 (B), and 10 (C) days post DLB inoculation. DLB cells can be seen in and

around leaf stomata (St) opening. (D) untreated plants 10 days post treatment.

Bacterial elongated rod shapes are indicated by white arrows.


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Figure 3: DLB cells located in the vascular tissues of carrot leaves and petioles.

Carrot plants were spray-inoculated with DLBGFP (A-E) or untreated (F), and

visualized under confocal microscopy 48 h post inoculation. (A) DLBGFP cells spread

across the surface of an unwashed carrot leaf. (B) DLBGFP cells located on the outer

surface of a carrot leaf cross-section. (C-D) Cross-section of two different carrot

petioles. DLBGFP cells are located in the xylem vessels and phloem cells. (E)

Longitudinal section of a washed petiole shows DLBGFP in a xylem vessel (indicated

by a white arrow). (St, stomata; Pc, palisade cells; Sc, spongy cells; Ep, epidermis;

Xy, xylem vessels; Ph, phloem tissue). Red fluorescence results from auto-

fluorescence of chloroplasts; green fluorescence from DLBGFP cells. All images are

merges of red auto-fluorescence, GFP fluorescence and bright-field microscopy.


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eSupporting information

Figure S1: DLB is present in Abraham’s balm and H. obsoletus environmental

samples. (A) DLB detected in environmental Abraham’s balm samples. Four samples

(1–8) from Abraham’s balm shrubs, two leaves per shrub, sampled in October. (B) H.

obsoletus samples (1 to 10) collected from Abraham’s balm shrubs in October. In

both gels (+) and (-) represent positive and negative controls, respectively; M, size

marker (500-bp band is indicated by an arrow) Expected size of the specific DLB

PCR product was ~400 bp.

Figure S2: DLBGFP colonization of a sesame plant following leaf inoculation.

Hoagland-grown 5-week-old sesame seedlings were inoculated by gentle brushing of

the first two true leaves with a DLBGFP soaked cotton swab. (A) DLB colonizes the

outer layers of sesame root (10 days post exposure). (B) Root cross-section showing

DLB colonization of inner-root cortex. Periderm (Pr), cortex (Co), Phloem (Ph), Xylem

(Xy). Green fluorescence was emitted by the DLBGFP strain. All images were

recorded with a confocal microscope.


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eTable 1: DLB detection in various plant species 5-7 days post application

†leaves were tested 14 days post inoculation without washing

Family Species (cV) Common name

Plant growth media

Inoculation method

Detection ratio

positive/total§

Malvaceae Gossypium Cotton Potting soil

Spray 5/5(a)

Potting soil

Drench 3/5(a)

Potting soil

Untreated 0/5(b)

Rutaceae Citrus sinensis

(Shamouti)

Orange Potting soil

Spray 4/5(a)

- Drench ND‡ Potting

soil Untreated 0/5(b)

Citrus grandis

Pomelo Potting soil

Spray 5/5(a)



Cucurbitaceae Cucumis melo

(Cezanne)

Melon Potting soil

Spray 10/10(a)

Potting soil

Drench 6/10(b)

Potting soil

Untreated 0/10(c)

Perlite Spray 10/10(a)

Perlite Drench 2/10(b)

Perlite Untreated 0/10(b)

Cucumis sativus

(Beit-Alpha)

Cucumber Potting soil

Spray 10/10(a)

Potting soil

Drench 2/10(b)

Potting soil

Untreated 0/10(b)

Perlite Spray 9/10(a)

Perlite Drench 3/10(b)

Perlite Untreated 0/10(c)

Solanaceae Nicotiana benthamiana

Benthamiana Potting soil

Spray 7/8(a)



Capsicum annuum

Pepper Perlite Spray 0/9(a)

Perlite Drench 1/9(a)


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‡ND- not-determined. §Different letters indicate statistical significance (α = 0.05) among application types within each plant species in the same medium.

Perlite Untreated 0/9(a)

Solanum tuberosum (Désireé)

Potato Perlite Spray 0/12(a)



Solanum lycopersicum (HA-29430)

Tomato Perlite Spray 0/12(a)



Apocynaceae Catharanthus roseus

Periwinkle Potting soil

Spray 9/9†(a)

Potting soil

Untreated 0/9(b)

Perlite Spray 0/9(a) Perlite Drench 5/9(b) Perlite Untreated 0/9(a)


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e Table 2: DLB penetration efficiency and persistence in carrot plants following foliar spraying and soil drenching inoculations Experiment I† Days after application 5 10 18 - Ratio of DLB positive plants following foliar spraying inoculation

12/12(a)

1/8(a)

2/12(a) -

Ratio of DLB positive plants following soil drenching inoculation

12/12(a)

1/8(a)

0/12(a)

-

Ratio of DLB positive plants in untreated plants 0/12(b)

0/8(a)

0/12(a)

-

Experiment II† Days after application 3 7 10 16 Ratio of DLB positive plants following foliar spraying inoculation

7/7(a)

7/8(a)

7/8(a)

3/8(a)

Ratio of DLB positive plants following soil drenching inoculation

3/5(b)

2/6(b)

0/7(b)

0/6(b)

Ratio of DLB positive plants in untreated plants 0/7(c) 0/8(c) 0/7(b) 0/6(b)†Different letters indicate statistically significant difference (α = 0.05) among application types within each sampling time point. Table 3: DLB penetration efficiency and persistence in sesame plants following foliar spraying and soil drenching inoculations. Experiment I – potting soil† Days after application 7 14 21 28 Ratio of DLB-positive plants following foliar spraying inoculation

10/10(a) 10/10(a) 6/10(a) 2/10(a)

Ratio of DLB-positive plants following soil drenching inoculation

0/10(b) 0/10(b) 0/10(b) 0/10(a)

Ratio of DLB-positive untreated plants

0/10(b) 0/10(b) 0/10(b) 0/10(a)

Experiment II – Hoagland agar† Days after application 7 14 21 28 Ratio of DLB-positive plants following foliar spraying inoculation

10/10(a) 10/10(a)

8/10(a) 2/10(b)

Ratio of DLB-positive plants following soil drenching inoculation

10/10(a) 9/10(a)

7/10(a) 3/10(ab)

Ratio of DLB-positive untreated plants

0/10(b) 0/10(b) 0/10(b) 0/10(b)

†Different letters indicate statistically significant differences (α = 0.05) among application types within each medium and sampling time point.


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