MICROSCOPY RESEARCH AND TECHNIQUE

Occurrence of Bacillus amyloliquefaciens as a Systemic Endophyte of Vanilla

Orchids

JAMES F. WHITE, JR.1*, MÓNICA S. TORRES1, RAYMOND F. SULLIVAN2,

RABIH E. JABBOUR3, QIANG CHEN1, MARIUSZ TADYCH1, IVELISSE

IRIZARRY1, MARSHALL S. BERGEN1, DAPHNA HAVKIN-FRENKEL1, FAITH C.

BELANGER1

1Department of Plant Biology and Pathology, Rutgers University, New Brunswick, New

Jersey, USA, Email: jwhite@aesop.rutgers.edu

2U.S. Army CBRNE Analytical & Remediation Activity, Aberdeen Proving Ground,

Maryland, USA

3U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground,

Maryland, USA

KEY WORDS lipopeptides, plant disease protection, defensive mutualism, endospores

ABSTRACT We report the occurrence of Bacillus amyloliquefaciens in vanilla orchids

(Vanilla phaeantha) and cultivated hybrid vanilla (V. planifolia x V. pompona) as a

systemic bacterial endophyte. We determined with light microscopy and isolations into

culture that tissues of V. phaeantha and the cultivated hybrid were infected by a bacterial

endophyte and that shoot meristems and stomatal areas of stems and leaves were densely

colonized. We identified the endophyte as B. amyloliquefaciens using DNA sequence

data. Since additional endophyte-free plants and seed of this orchid were not available,

additional studies were performed on surrogate hosts Amaranthus caudatus, Ipomoea

tricolor and I. purpurea. Plants of A. caudatus inoculated with B. amyloliquefaciens

demonstrated intracellular colonization of guard cells and other epidermal cells,

confirming the pattern observed in the orchids. Isolations and histological studies suggest

that the bacterium may penetrate deeply into developing plant tissues in shoot meristems,

forming endospores in maturing tissues. We demonstrated that B. amyloliquefaciens

produced fungal inhibitors in culture. Further, in controlled experiments using morning

glory seedlings, we demonstrated that the bacterium promoted seedling growth and

reduced seedling leaf necrosis due to pathogens. We detected the gene for

phosphopantetheinyl transferase (sfp), an enzyme in the pathway for production of

antifungal lipopeptides, and purified the lipopeptide ‘surfactin’ from cultures of the

bacterium. Based on our data, we hypothesize that B. amyloliquefaciens is a robust

endophyte and defensive mutualist of vanilla orchids. Whether the symbiosis between

this bacterium and its vanilla hosts can be managed to protect vanilla crops from diseases

is a question that should be evaluated in future research.

INTRODUCTION

There is growing evidence that plants in natural populations are inhabited by non-

pathogenic microbes (Stone et al., 2000; Compant et al., 2005; Johnston-Monje and

Raizada, 2011). Microbial components of the plant microbiome are both bacterial and

fungal and may exist on plant surfaces and interiors (James, 2000; Angus and Hirsch,

2013; Arnold and Lutzoni, 2007; Reinhold-Hurek and Hurek, 2011). Although we do not

thoroughly understand how most non-pathogenic microbes interact with plants or with

one another, we do have several examples. For instance, research on endophytic fungi

demonstrates that they may enhance host plant resistance to biotic and abiotic stresses,

thus protecting plants from herbivory, diseases or environmental stresses such as soil

heavy metals, drought, or extreme temperatures (Redman et al, 2002; Clarke et al., 2006;

Hamilton et al., 2012; White and Torres, 2012; Schardl et al., 2013).

Vanilla orchids are hemi-epiphytic vines that climb trees and often become fully

epiphytic through loss of soil roots (Bayman et al., 2011). Currently, vanilla bean

production worldwide is threatened by an epidemic of fungal root rot diseases that is

projected to curtail supply of vanilla in coming years (Bayman et al., 2011). Due to

economic considerations, pesticides to reduce losses are not generally used in production

of vanilla beans (Bayman et al., 2011). In an effort to identify defensive bacterial

endophytes that might eventually be used to control diseases and support growth in

cultivated vanilla plantations we initiated studies on vanilla endophytes.

MATERIALS AND METHODS

Plant Materials and Survey of Vines

A single plant of hybrid vanilla (V. planifolia x V. pompona) was obtained from

naturalized populations in Puerto Rico. A single plant of Vanilla phaeantha was obtained

from a commercial source. From these two original plants numerous individual clones

were produced through fragmentation of vines. This was done by cutting vines into

segments that were approximately 1m in length and new roots and shoots were generated

on vines. In addition, five plants of V. planifolia were obtained from a commercial source

and confirmed Bacillus-free via failure to isolate from leaves. To survey for Bacillus

endophytes in natural populations of V. phaeantha, populations of the orchid were

located in Fakahatchee Strand Preserve State Park in South Florida. Because plants of V.

phaeantha are protected, sampling for the bacterium was done non-destructively. To

sample for Bacillus in plants, heat sterilized cotton applicators were rubbed into fluid

visible in the shoot tips of V. phaeantha plants, then placed into sterile tubes for transport

to the laboratory 3 to 5 days later. Cotton applicators were pasteurized to eliminate non-

endospore formers by heating applicators to 50ºC for two hours. Pasturized cotton

applicators were rubbed onto the surface of 10% Tripticase Soy Agar (TSA; Difco)

plates, incubated at laboratory ambient temperature and examined after 1 week for the

characteristic white, dry colonies of B. amyloliquefaciens with abundant endospores

(Figs. 1 and 2).

Isolation of Bacteria from Plants

For initial isolations of B. amyloliquefaciens from plants (three plants each of V.

phaeantha and hybrid vanilla), shoot tips, leaves and roots down to five nodes from the

shoot tip and 5cm stem segments from each internode down to the fourth internode were

removed from plants. All tissues were surface disinfected in 4% sodium hypochlorite

solution for 30 min with constant agitation, followed by agitation in sterile water for 10

min. Plant tissue segments were then excised into pieces approximately 2-3 mm2 and

plated onto 10% TSA (three tissue pieces/plate; and 5 plates per tissue sample), then

incubated for seven days at laboratory ambient temperature.

To access penetration of bacteria into stem tissues a five mm diam cork borer was

heat sterilized and used to core the center from six cm-long stem segments (surface

disinfected as above) cut from the third internode from the stem tips of five plants of V.

phaeantha. Stem cores were then rinsed in sterile water and cut into pieces 2-3 mm diam;

then fifteen pieces were plated onto 10% TSA medium and incubated for seven days at

laboratory ambient temperature. After incubation plates were examined to determine the

percentage of tissue pieces showing B. amyloliquefaciens growth (Table 1).

Identification of Bacteria

For bacterial identification, ~900 base pairs of the 16S rDNA region were

obtained (Baker et al., 2003). Sequences were compared to sequences available in the

NCBI GenBank database to identify the closest matches. For amplification of the

phosphopantetheinyl transferase (sfp) gene a 594bp fragment was amplified by PCR

using two primers, sfp-f (5’-ATGAAGATTTACGGAATTTA-3’) and sfp-r (5’-

TTATAAAAGCTCTTCGTACG-3’) (Hsieh et al., 2004). The amplification was

conducted using a program of denaturing at 94oC for 1 min, annealing at 46oC for 30 s

and extension at 72oC for 1 min, for a total of 36 cycles. The PCR product was analyzed

by 1% agarose gel electrophoresis and then sent for sequencing. Both sequences were

submitted to GenBank (16S rDNA = KF765481; sfp gene = KF765482).

Histology of Infected Plants

To visualize bacteria in biofilms of vanilla shoots (Figs. 3-7) and epidermal cells

of vanilla orchid tissues were stained with the nucleic acid stain SYTO®9/Propidium

Iodide provided in the Live/Dead Bacterial Viability Kit (Life Technologies, Carlsbad,

CA) and observed using fluorescence microscopy on a Zeiss Axioskop® using the filter

system for DNA excitation (485⁄498 nm for Fig. 4). To visualize the process of tissue

entry in vanilla and Amaranthus tissues, confocal microscopy was employed using the

nucleic acid stain SYTO13® (Life Technologies, Carlsbad, CA). Tissues were examined

using a Zeiss LSM 710® confocal microscope using lasers 48 (for nucleic acids) and 633

nm (for chlorophylls) with a 100x objective (Figs. 5,6, 11-14). To make 3-D

reconstructions (Figs. 11, 12 and 14), images were created using the z-stack application

in the Zen® software package. Images were constructed using slices through the upper

epidermal cell layer of tissues (24µm @ 0.3µm section intervals; 84 sections).

To visualize bacteria on the surfaces of young rapidly growing vanilla leaves

bordering the shoot tip, thin free-hand sections of epidermal layers were made using a

razor. These were then stained using safranin-o (0.1% aqueous) at room temperature for 5

min at ambient laboratory temperature (Fig. 7). To stain endospores in older leaf tissues,

longitudinal sections were stained using the endospore specific stain malachite green

(0.5% aqueous) for 15 min at 60C. Sections were washed with several changes of water

for 10 minutes by moistening tissues with water then blotted dry until no more green

coloration emerged from tissues (Gerhardt et al., 1994). Tissues were counterstained with

safranin-o as above, then examined with light microscopy (Figs. 8-10; Gerhardt et al.,

1994). To visualize bacteria within tissues of Vanilla phaeantha, leaves and stem cores

were cut into 1 x 3mm pieces and fixed in FAA fixative for 24 hours, then dehydrated

and embedded in LR White® Acrylic resin. Sections 1 µm -thick were made using glass

knives and stained on a slide warmer for 30 seconds using safranin-o (0.1% aqueous),

rinsed, and counterstained for 30 seconds using toluidine blue (0.1% aqueous; Gerhardt et

al., 1994). Sections were visualized using light microscopy (Figs. 15-17).

Amaranthus caudatus Infection Experiment

To evaluate the colonization of plant tissues by B. amyloliquefaciens an

inoculation experiment was conducted using Amaranthus caudatus. Seeds of A. caudatus

were surface disinfected using 4% sodium hypochlorite for 10 mins with constant

agitation, then washed for 5 mins in sterile distilled water. Twenty-five seeds were then

placed on each of 10 Petri dishes containing moist filter paper. One ml of a suspension of

B. amyloliquefaciens (concentration OD 600 nm = 0.5) was then added to five of the

plates, and one ml of sterile water was added to the other five plates. Plates were sealed

in clear plastic zip-lock bags and incubated on a lab bench for 10 days, after which both

treatments were examined and seedling root lengths measured and seedlings weighed.

Evaluation of Endospore Dormancy and Isolation Frequency

To evaluate the extent to which endospore dormancy within tissues of vanilla

vines reduces isolation frequency from mature plant tissues, we conducted a bacterial

isolation experiment using media: 1.) alanine-glucose agar (1% L-alanine: 1% glucose:

1.2% Agar Noble); and 2.) 10% TSA (0.4% Tripticase Soy Agar, Difco: 1.2% Agar

Noble). In addition, to trigger endospore germination some of the tissues were heated for

two hours in an oven at 60ºC prior to isolation from plant tissues. Plant tissues were

surface disinfected as previously described and cut into 2 mm2 pieces. Five Petri plates of

each treatment were prepared with three tissue pieces on each plate. Plates were

incubated for seven days in laboratory ambient conditions prior to examination for

presence of B. amyloliquefaciens and results recorded in Table 2.

Antibiosis Experiments

To examine in vitro production of antifungal compounds B. amyloliquefaciens

was streaked onto 10% TSA plates in a line. Plugs 7 mm in diam of fungi Alternaria

alternata, Colletotrichum dematium, Fusarium oxysporum and Lasiodiplodia theobromae

were placed on either side of the bacterial line at a distance of 2 cm from the bacterial

streak. Fifteen replicates were made for each fungus and the plates were incubated at lab

ambient temperature for ten days, after which zones of inhibition between bacterial

streaks and fungal colonies were measured (Table 3).

Experiments Using Morning Glory Seedlings

In the first experiment seeds of Ipomoea tricolor (cultivar Blue star) were treated

by soaking for 24 hours in sterile water or in a suspension of B. amyloliquefaciens

(concentration OD 600 nm = 0.5). Seeds were then placed into Petri plates (ten seeds

/plate and five plates/treatment) containing sterile moist filter paper and incubated in

Ziplock® plastic bags in laboratory ambient temperature for ten days, after which plates

were examined and seedlings assessed for effects on growth (Fig. 18). In a second

experiment seeds of Ipomoea purpurea (cultivar Crimson rambler) and Ipomoea tricolor

(cultivar Blue star) were soaked in a suspension of B. amyloliquefaciens or sterile water

as above, then planted in a flat containing potting mix comprised of three parts peat moss

to two parts perlite. Twenty-five seeds were planted in rows with each treatment/variety

forming a single row. The flat was then placed under a mister to maintain high moisture

around seedlings during the germination process. After two weeks twenty seedlings of

each treatment were harvested and the cotyledon leaf area was estimated (leaf length x

width). The percentage of seedlings in each treatment showing necrotic lesions was also

determined. These data are summarized in Table 4.

Fungal Inhibitor Purification and Identification

Bacillus amyloliquifaciens lipopeptides were produced in a liter of 10% tryptic

soy broth inoculated with 5 ml of an overnight culture of the bacterium and incubated for

24 hrs at 30°C shaking at 150 rpm. The bacterium was pelleted and the pH of the

supernatant was reduced to 3 with concentrated acetic acid and further reduced to pH 2

with concentrated hydrochloric acid. The precipitate from the acidified supernatant was

collected by centrifugation and resuspended in 45 ml of butanol. Butanol insoluble

material was pelleted by centrifugation and further removed by a 0.22 µ syringe filter.

The sample was introduced into the Thermo Scientific LTQ XL Linear Ion Trap Mass

Spectrometer by autosampler injection of 10 ml of the sample into a 100 ml min-1 HPLC

flow of methanol:water:formic acid (50:49.9:0.1) buffer without chromatographic

separation. The APCI-MS was tuned in positive mode to the 1036.7 m/z analyte with a

vaporizer temperature of 450°C, a capillary temperature of 200°C and an 80/20 N2 sheath

gas/auxillary gas flow rate. Collision induced dissociation energy of 50% was used to

produce the most effective fragmentation.

Toothpick Inoculation Experiment

Wooden toothpicks were sterilized by autoclaving in a 1% sucrose solution.

Sterilized toothpicks were then placed on the surface of 10% TSA plates that were

inoculated with plugs of Alternaria alternata. After fungal mycelium colonized

toothpicks, they were removed and dipped in a suspension on B. amyloliquefaciens

(concentration 600 nm OD = 0.5) or sterile water. Twenty toothpicks for each treatment

were then used to wound leaves of greenhouse maintained plants of V. planifolia. After

ten days the diameters of necrotic zones around toothpicks were measured.

RESULTS

Preliminary Survey

A preliminary survey of plants of Vanilla planifolia (4 plants), V. pompona (2

plants), a hybrid vanilla [V. planifolia x V. pompona] (6 plants) and V. phaeantha (10

plants) demonstrated presence of a bacterial endophyte in aerial tissues of vines of the

hybrid vanilla and the North American native species V. phaeantha. A white irregular

bacterial colony that produced masses of ellipsoidal to cylindrical refractive endospores

was consistently isolated on 10% TSA from surface sterilized pieces of shoot meristems,

stems and leaves of all plants of both the hybrid vanilla and V. phaeantha (Figs. 1 and 2).

Natural populations of V. phaeantha in Fakahatchee Strand Preserve State Park near

Naples, Florida were also sampled and found to also contain B. amyloliquefaciens in 8 of

14 plants sampled.

Bacterial Identification

For bacterial identification, the 16S rDNA and the phosphopantetheinyl

transferase (sfp) genes were sequenced. The vanilla endophyte sequence for the 16S

rDNA sequence (NCBI Accession KF765481) was similar to B. amyloliquefaciens strain

FZB42 (NCBI Accession NR_075005.1), a strain commonly used in biocontrol products.

The sequence for the sfp gene (GenBank Accession KF765482) was identical to the sfp

gene in B. amyloliquefaciens strains E1PA (EMBL-EBI Accession KC711052.1).

Phylogenetic trees constructed using our sequences demonstrated placement in the

species B. amyloliquefaciens for both sequence regions (trees not shown).

Observations on Shoot Tips

Examination of shoot meristems showed presence of liquid exudate from all

plants shown to be infected by B. amyloliquefaciens (Fig. 3). Microscopic examination of

primordial leaf tissues in the shoot apex revealed presence of bacterial biofilms

containing rods and coccoid forms (Fig. 4). Bacterial biofilms were seen to form over

epidermal cell junctions in differentiating leaves of shoot tissues (Fig. 5). Bacteria were

observed to densely colonize cells associated with stomatal openings, especially guard

cells and adjacent epidermal cells (Figs. 6-10; also 11-14).

Histological Studies and Distribution of Bacillus in Plant Tissues

Using confocal microscopy bacteria were observed to accumulate at cell

junctions of primordial leaves (Figs. 5 and 6). Examination of young leaves bordering the

shoot tip showed that bacteria also accumulate around guard cells and subsidiary cells of

stomata (Figs. 5-7). An examination of older leaves shows an abundance of endospores

characteristic of B. amyloliquefaciens on the surface of and within guard cells and

subsidiary cells (Figs. 8 and 9). In addition, endospores were occasionally seen in clusters

within epidermal cells (Fig. 10). In some slide preparations endospores were seen to fill

stomatal cavities (Fig. 9). Sections through vascular bundles of leaves also show rods that

resembled those of B. amyloliquefaciens in xylem, phloem and parenchyma cells but

endospores were not evident (Figs. 15-17).

Isolations of B. amyloliquefaciens from cores from the center of stems confirm

that the bacterium penetrated deeply into stem tissues (Table 1). Mature vanilla plant

tissues that were heat pre-treated prior to isolation of bacteria showed a significant

increase in isolation frequency of B. amyloliquefaciens into alanine-glucose agar (Table

2).

Confirmation of Intracellular Penetration by B. amyloliquefaciens

In Amaranthus caudatus inoculation experiments B. amyloliquefaciens was

observed to colonize leaf tissues in a comparable way to that observed in vanilla.

Numerous cells of the bacterium were seen to enter guard cells and were often seen close

to chloroplasts (Figs. 11 and 12). In other epidermal cells bacteria were intercellular, but

remained in the periphery of the cell in cytoplasm (Fig. 14). Biofilms of bacteria formed

on the leaf surface over cell junctions (Fig. 13). Un-inoculated control plants did not

show biofilm formation on seedling leaves, and intracellular bacteria were not observed

in confocal microscopic observation of the plant tissues. Amaranthus seedlings colonized

by B. amyloliquefaciens had seedling leaves that were brighter red in color and smaller

than in the water treated controls. Bacterial colonized seedlings were on average smaller

(seedling wet wt = 5.19 ± 0.79 mg vs 7.82 ± 1.1 mg (mean ± standard deviation) for

water controls). Seedlings colonized by B. amyloliquefaciens also had notably shorter

roots than controls (10.9 ± 3.48 mm vs 25.45 ± 4.74 mm for water controls).

Fungal Inhibitors and Disease Control

Isolates of B. amyloliquefaciens from both V. phaeantha and the hybrid vanilla

were found to produce fungal inhibitors in culture (Table 3). We further amplified the

phosphopantetheinyl transferase (sfp) gene for iturin A production from the V. phaeantha

isolate. The supernatant from a liquid culture of B. amyloliquefaciens was acid

precipitated and analyzed. After Atmospheric Pressure Chemical Ionization Mass

Spectrometry (APCI-MS), a prominent 1036.7 (M+H)+ ion (Fig. 19) was further

investigated by MS/MS using resonance excitation collision induced dissociation (CID).

Although CID resulted in highly efficient precursor ion fragmentation, it was subject to a

low mass cutoff and did not allow trapping of fragment masses below 27.5% of the

precursor mass. Pulsed Q Collision Induced Dissociation (PQD) was attempted to

observe predicted low m/z fragments but very few ions of any m/z were observed and no

low m/z ions were observed (data not shown). Nevertheless, observed ions with m/z

>285 convincingly showed the 1036.7 m/z ion to be C15 surfactin, a powerful surfactant

lipopeptide compound produced by various Bacillus spp. (Fig. 20). In addition, common

morning glory seedlings derived from seeds treated with B. amyloliquefaciens showed

significantly larger seedling leaves (cotyledons) than those of water treated controls;

bacterial treated seedlings demonstrated a lower incidence of fungal lesions/necrosis than

seedlings without bacteria (Table 4). Microscopic examination of the cotyledons of

morning glory seedlings treated with the bacterium (see Fig. 18) revealed dense bacterial

biofilms on seedling leaves (Fig. 13), while water treated controls lacked any evidence of

biofilms on seedling leaves. In experiments using Bacillus-free Vanilla planifolia leaves

wounded using toothpicks bearing the fungal pathogen Alternaria alternata, wounds

created using toothpicks bearing only the fungus had a greater lesion diameters after a 4-

day incubation period than leaves wounded using toothpicks treated with the fungal

pathogen and B. amyloliquefaciens (fungus only lesion diam = 7.1538 ± 0.67791 mm

(mean ± standard deviation); fungus + bacterium lesion diam = 2.84615 ± 0.93264 mm;

N = 15; a T-test for paired samples analysis gave p < 0.01).

 

DISCUSSION

This study provides evidence that Bacillus amyloliquefaciens is both epiphytic

and endophytic in vanilla plants. While not exhaustively examined, the isolation of the

bacterium from tissues of the hybrid vanilla from Puerto Rico and plants of V. phaeantha

in South Florida suggest that B. amyloliquefaciens may be a widespread symbiont of

vanilla orchids. The fact that we did not isolate it from several plants of Vanilla planifolia

and V. pompona may be an indication that it is not present in all vanilla orchids.

Histological studies on V. phaeantha leaf tissues show that bacteria form biofilms

in shoot tips on leaf and stem primordia. These biofilms concentrate at epidermal cell

junctions and over stomata. This may be due to nutrient leakage from stomata and cell

junction areas. Based on observations made using vanilla plants and the surrogate host

Amaranthus caudatus, intracellular infection of plant tissues appears to occur beneath

these biofilms. In the process of infection rods appear to convert into spherical cells, as

observed in the infection of seedling leaves of Amaranthus caudatus (Figs. 11-14). These

spherical bacterial cells are likely cell wall deficient bacteria, often called L-forms.

Similar L-forms have been shown to occur in Bacillus subtilis and many other bacteria

when they enter into the cytoplasm of eukaryotic cells (Leaver et al., 2009). L-form

development is accompanied by reduced virulence and long-term persistence in the host,

thus bacterial L-forms of pathogenic bacteria have been considered to be symbiotic or

latent phases (Amijee et al., 1992; White et al., 2014). Based on the position of bacteria

in epidermal cells, the L-forms appear to be limited to the cytoplasm (Figs. 11 and 12).

Guard cells may be rich in nutrients and cytoplasmic contents due to presence of

chloroplasts there. The presence of bacteria around the margins of other epidermal cells

(Figs. 11 and 14) may be due to the large central vacuole in those cells. We did not

observe entry of B. amyloliquefaciens into the vacuole but rather it remained in the

cytoplasmic component of cells.

It seems likely that entry of the bacterium into cells of the plant is accomplished

through use of cell wall degrading enzymes. Confocal microscopy of the inner top walls

of two guard cells (Fig. 12) shows the bacteria in guard cells, and surrounding the

bacteria are darker halos that may represent softened or altered plant cell walls. Other

symbiotic bacteria are also thought to enter plant cells using cell wall degrading enzymes

(see Kovtunovych et al., 1999; Compant et al., 2005).

Entry of B. amyloliquefaciens into guard cells and subsidiary cells could affect the

functionality of stomata. Even if guard cells continued to function after entry of bacteria,

the eventual proliferation of bacteria in the stomatal chambers could prevent their closure

or reduce gas exchange with the exterior (Fig. 9). It is not clear to what extent

functionality of stomata is affected by the bacterial endophyte. However, if stomatal

functioning is reduced, B. amyloliquefaciens could be considered a weak pathogen of

vanilla orchids. Along these lines, in inoculation experiments involving Amaranthus

caudatus we recorded reductions in size and weight of seedlings as a result of infection

by B. amyloliquefaciens. This reduced growth could be a reflection of a parasitic

interaction between the bacterium and Amaranthus and could be a further indication of

the cost of the interaction to vanilla plants (Cheplick, 2007).

Two lines of evidence suggest that B. amyloliquefaciens penetrates vanilla plant

tissues but very rapidly goes dormant in most tissues. We observed abundant endospore

formation in leaf epidermal cells (Figs. 8-10). In addition, heat pre-treatments of mature

plant tissues prior to isolation attempts on alanine-glucose agar increased the rate of

isolation from tissues. Because bacterial endospores are heat activated (Keynan et al.,

1964), enhanced isolation of the bacterium from tissues after heat treatments is consistent

with a scenario where bacteria are present as endospores in plant tissues.

It is unclear how deeply B. amyloliquefaciens penetrates into vanilla orchid

tissues. We have confidence that the bacterium penetrates into epidermal layers because

we observed rods and endospores characteristic of the bacterium in these tissues, and our

infection experiments using Amaranthus caudatus confirmed at least superficial infection

into epidermal cells. However, we isolated the bacterium from deep within stem cores

where epidermal layers had been removed, suggesting that the tissues of the vanilla

orchid may be permeated in all parts by the bacterium. In sections of stem vascular

bundles we were able to visualize aggregations of bacterial rods in xylem, phloem and

parenchyma cells (Figs. 15-17). However, because we did not observe endospores

forming within tissues of vascular tissues, and our stains were non-specific, we cannot be

sure that they belong to B. amyloliquefaciens. Regardless of this uncertainty, the

consistent isolation of this bacterium from internal tissues of the stem shows that it

penetrates deeply into plant tissues. In addition, since endospores generally form as a

result of nutrient restriction (Keynan et al., 1964), it seems reasonable that the availability

of a continuous supply of sugars and other nutrients within vascular tissues of vanilla

vines reduces tendency of the bacterium to form endospores and could account for lack of

any evidence of endospores in vascular tissues (Figs. 15-17).

Vertical dissemination of the bacterium is facilitated by growth of the bacterium

within shoot tips of vanilla orchids that enables it to colonize all primary leaves and

stems as they are formed. Any new stems arising from axillary buds and aerial roots also

become colonized by the bacterium. Thus the bacterium is able to move from older stems

to newly initiated meristems. It is possible that this movement is due to bacterial

movement in vascular tissues that connect all parts of the plant. Alternatively, movement

of bacteria from older stems to newly initiated meristems may be due to surface

movement of bacteria on plants, since bacteria may move using swarming motility even

when surface plant moisture is lacking. Although, how B. amyloliquefaciens moves

within plants is unknown, it is certain that bacterial colonization of new stems enables the

bacterium to transmit to new clonally produced plants. In vanilla orchids clonal

propagation occurs frequently with most new plants arising from fragmentation of

parental vines. In agricultural production vines are broken into segments to initiate new

vines (Bayman et al., 2011). In our greenhouse culture of the hybrid vanilla and V.

phaeantha we produced dozens of new plants using fragmented vines and in every case

plants retained B. amyloliquefaciens. We were not able to evaluate whether the bacterium

would vector through seeds since our plants did not produce fruits.

Liquid accumulation in shoot tips of infected vines suggests another mechanism

for horizontal transmission of the bacterium to uninfected plants. The bacterium could

move horizontally between plants via insects that feed on the shoot meristem liquid. We

were able to repeatedly isolate B. amyloliquefaciens from shoot apex liquid. Insects, such

as flies, may vector bacteria to other plants when they feed or drink from the liquid in the

shoot tip. It is equally possible that bacteria that flow out of stomata may be dispersed by

a rain splash mechanism to adjacent plants. The high concentrations of bacteria that we

observed associated with the surfaces of vanilla plants makes this water splash

mechanism highly probable. However, additional work is needed to determine whether

either of these proposed dissemination mechanisms are important in natural populations.

Beneficial effects of infection by B. amyloliquefaciens on infected vanilla plants

is difficult to prove because all of our hybrid and V. phaeantha plants contained the

bacterium and controlled experiments using vanilla orchids were not possible. However,

we were able to demonstrate that B. amyloliquefaciens secretes surfactin, a lipopeptide

composed of a 7 amino acid cyclic ring and an alkyl tail of varied length and structure

(Raaijmakers et al., 2010). Surfactins have demonstrated fungitoxicity towards

Magnaporthe grisea, Sclerotinia sclerotiorum, and Fusarium verticillioides (Tendulkar et

al., 2007; Ongena and Jacques, 2007; Snook et al., 2009; Alvarez et al., 2012). Induction

of host plant systemic resistance by surfactins has also been demonstrated through

stimulation of the lipoxygenase pathway (Ongena and Jacques, 2007). Surfactin

lipopeptides have been shown to reduce bacterial colonization of plants by inhibition of

bacterial motility and biofilm formation (Chowdhery et al., 2013). Because of the

capacity to suppress root and foliar diseases in plants, several strains of B.

amyloliquefaciens have been employed in biocontrol formulations of commercial

products (Ongena and Jacques, 2007). In our experiments we found that the vanilla

Bacillus demonstrated inhibitory antagonism toward several fungal species when cultured

together. In another experiment using morning glory seedlings with and without the

bacterium, we found that the bacterium promoted seedling growth and reduced seedling

leaf lesions, probably due to damping off fungi (Fig. 18; Table 4). While not definitive,

we have enough evidence to hypothesize that B. amyloliquefaciens is a naturally

occurring defensive mutualist of vanilla orchids (Clay, 1988; Elliot et al., 2000).

Validation of the defensive nature of the vanilla Bacillus will require controlled

experiments with Bacillus-infected and -free vanilla plants. Whether, this bacterium has

potential to control diseases that are currently affecting vanilla orchids (Vanilla planifolia

and V. pompona) in commercial operations will require additional evaluation.

CONCLUSIONS

We present evidence that B. amyloliquefaciens is a common systemic endophyte

of some species of vanilla orchids. This is the first plant where it has been shown that

growth-promoting bacteria colonize plants through shoot tissues rather than root tissues.

We further show that the bacterium colonizes plant cells intracellularly. This has not been

shown in many other plants and where it has been shown, cells invaded have been root

cells (Paungfoo-Lonhienne et al., 2010; White et al., 2014). We demonstrated that B.

amyloliquefaciens contains genes for and produces antifungal lipopeptides; and that

application of the bacterium to morning glory seeds promoted seedling growth and

reduced disease. On the basis of the studies reported here we hypothesize that B.

amyloliquefaciens is a widespread defensive mutualist of some species of vanilla orchids.

Figs. 1‐2. Bacillus amyloliquefaciens. 1. Vanilla stem segments showing characteristic colonies of the bacterium emerging from plant tissues on 10% TSA medium. 2. Densely‐stained endospores (arrows) and longer, lightly‐stained, vegetative cells (stained with malachite green endospore stain).  

Figs. 3-6. Vanilla phaeantha bearing Bacillus amyloliquefaciens. 3. Shoot tip showing

liquid exudate (arrow) at shoot opening of infected plants. 4. Biofilm on primordial leaf

in shoot showing rods (arrow) stained with SYTO9®. 5. Confocal image of surface of

leaf in shoot tip showing bacteria (arrows) in biofilms at cell junctions stained with

SYTO13®. 6. Confocal image showing bacteria (arrows) on and around guard cells

stained with SYTO13®.

Figs. 7-10. Vanilla phaeantha. 7. Bacterial accumulation (arrows) around stoma on primordial leaf bordering shoot tip, stained with safranin (0.1%, aqueous). 8. Bacterial endospores (arrows) on guard cells of an older leaf, stained with endospore stain malachite green (0.1%, aqueous) followed by safranin. 9. Bacterial endospores (arrows) in a substomatal chamber of an older leaf, stained with endospore stain malachite green (0.1%, aqueous) followed by safranin. 10. Clusters of endospores (arrows) within an epidermal cell of an older leaf, stained with malachite green.

Figs. 11, 12, 14. Confocal microscopy of Bacillus amyloliquefaciens in a seedling leaf of Amaranthus caudatus stained with SYTO13. 11. Image showing spherical bacterial L-forms (orange; arrows) within epidermal cells. White arrows indicate bacterial L-forms in guard cells (chloroplasts are blue). Black arrows indicate spherical bacterial L-forms in adjacent epidermal cells. 12. Maximum intensity projection showing spherical bacterial L-forms (arrows) within guard cells near chloroplasts (red). 13. Surface of morning glory seedling leaf showing bacterial biofilm (arrows) after seeds were treated with B. amyloliquefaciens. 14. Maximum intensity projection using a subset of z-stack showing bacterial accumulations at periphery of epidermal cells (arrows).

Figs. 15-17. Bacteria in vascular tissues of Vanilla phaeantha. 15. Section through vascular bundle in stem showing bacteria (arrows) in xylem (stained with safranin and counterstained with toluidine blue). 16. Transverse section of vascular bundle in stem showing bacteria within xylem (arrows; stained with safranin). 17. Cross section of xylem vessel showing bacterial biofilm (arrow) adhering to inner xylem wall (stained with safranin). 18. Morning glory seedlings (ten days old) showing seedlings with unexpanded cotyledons resulting from water treated seeds (A) and faster growing seedlings with expanded cotyledons resulting from B. amyloliquefaciens-treated seeds (B).

 

Fig. 19. APCI mass spectrum of the precipitated supernatant from an overnight culture of

the Bacillus amyloliquifaciens endophyte. Annotated peaks correspond to surfactins with

various fatty acid tail (C) lengths and charged ions.

Fig. 20. APCI-MS/MS spectrum (averaged from 31 spectra) of the 1036.7 m/z precursor

ion using collision induced dissociation (CID) (50% collision energy). The upper right

box lists predicted fragment ions with observed ions underlined and labeled in the

averaged spectrum. The structure of Surfactin A is shown (upper left).

TABLE 1. Isolation of B. amyloliquefaciens from epidermal tissues and stem cores of V.

phaeantha onto TSA medium1

Tissue Vine number

1 2 3 4 5

Epidermis 91.67% 100% 100% 100% 0%

Core 100% 88% 100% 100% 83.3%

1Percentage of pieces showing emergence of bacterium (N=15)

TABLE 2. Effect of medium and heat treatment on isolation frequency

Tissue Pre-treatment Alanine-glucose

agar

10% Trypticase Soy

Agar

Shoot tip No heat 100% 100%

Leaf 1 No heat 0% 100%

Leaf 2 No heat 20% 100%

Leaf 3 No heat 0% 100%

Leaf 4 2 hr @ 60C 100% 100%

Leaf 5 2 hr @ 60C 73.33% 100%

Leaf 6 2 hr @ 60C 0% 0%

Stem No heat 40% 86.67%

Stem 2 hr @ 60C 78.57% 93.33%

TABLE 3. Fungal pathogen inhibition by B. amyloliquefaciens in culture1

Fungal Pathogens

Plant Source Fusarium

oxysporum

Lasiodiplodia

theobromae

Colletotrichum

dematium

Alternaria

alternata

Hybrid vanilla 4.8 ± 1.3 5.8 ± 0.92 11.4 ± 1.26 11.4 ± 0.97

V. phaeantha 3.8 ± 0.83 7.7 ± 1.34 10.9 ± 2.85 9.2 ± 1.4

1 Data is mean ± standard deviation of inhibition zone diameters in mm (N = 20)

TABLE 4. Bacillus amyloliquefaciens effects on morning glory seedling cotyledon size

and necrosis

Cultivar Water control B. amyloliquefaciens

Leaf area

(mm2)

Percent

necrotic

Leaf area (mm2) Percent

necrotic

Crimson

rambler

251.9 ± 92.21 90% 483.3 ± 132.1 0%

Blue star 435.4 ± 240.87 100% 742.5 ± 448.4 0%

1Significant difference for both varieties when water controls are compared to bacterial

treatments (Student T test; P < 0.05; N=20).

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*Correspondence to: James F. White, Department of Plant Biology and Pathology,

Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901, USA. E-mail:

white@rci.rutgers.edu. Received ____________2014;

REVIEW EDITOR: Professor Alberto Diaspro.

Contract grant sponsor: USDA NIFA Multi-State Project 3147, the John E. and Christina

C. Craighead Foundation, and the New Jersey Agricultural Experiment Station. The

authors are grateful to Dr. Maurine Bonness and Mike Owen for assisting with sampling

plants of V. phaeantha in cypress swamps of the Fackahatchee Strand Preserve State Park

in South Florida.