ORIGINAL PAPER

Heat treatment induced bacterial changes in irrigation waterand their implications for plant disease management

W. Hao • C. X. Hong

Received: 22 August 2013 / Accepted: 12 December 2013

� Springer Science+Business Media Dordrecht 2013

Abstract A new heat treatment for recycled irrigation

water using 48 �C for 24 h to inactivate Phytophthora and

bacterial plant pathogens is estimated to reduce fuel cost

and environmental footprint by more than 50 % compared

to current protocol (95 �C for 30 s). The objective of this

study was to determine the impact of this new heat treat-

ment temperature regime on bacterial community structure

in water and its practical implications. Bacterial commu-

nities in irrigation water were analyzed before and after

heat treatment using both culture-dependent and -inde-

pendent strategies based on the 16S ribosomal DNA. A

significant shift was observed in the bacterial community

after heat treatment. Most importantly, bacteria with bio-

logical control potential—Bacillus and Paenibacillus, and

Pseudomonas species became more abundant at both 48

and 42 �C. These findings imply that the new heat treat-

ment procedure not only controls existing plant pathogens

but also may make the heat-treated irrigation water a more

antagonistic environment against plant pathogens, pro-

moting sustainable disease management.

Keywords Biological control activity � Bacillus �Colony PCR–SSCP � Paenibacillus � PCR–DGGE �Pseudomonas

Introduction

Numerous plant pathogens, including zoosporic organisms,

bacteria, viruses, fungi, and nematodes, have been found in

irrigation water (Thomson and Allen 1974; Geldreich

1996; Hong and Moorman 2005; Cayanan et al. 2009).

These waterborne pathogens pose a serious threat to a

variety of agriculturally important crops. Among the most

common and destructive plant pathogens in irrigation water

are those belonging to the genera Pythium Pringsh and

Phytophthora de Bary (Baker and Matkin 1978; Stang-

hellini and Rasmussen 1994; Hong and Moorman 2005).

Bacteria are also common pathogens found in waters (Toze

1999, 2006). Therefore, water treatment is very important

to reduce potential crop losses caused by these pathogens.

Heat pasteurization is one of the safest and most reliable

methods for water decontamination in agriculture (Runia

1995; van Os 1999). The current heat treatment recom-

mends raising and maintaining water temperature at 95 �C

for 30 s (Runia et al. 1988; McPherson et al. 1995). This

technology is widely used in the Netherlands and the

United Kingdom by greenhouse growers but to lesser

degree in other countries including the US due to its energy

cost and environmental footprint.

Our recent study revealed that water treatment temper-

ature can be lowered to 48 �C with extended exposure time

to eliminate Phytophthora and several pathogenic bacterial

species, including Agrobacterium tumefaciens, Erwinia

carotovora, E. amylovora, Pseudomonas syringae

pv. tomato, Ralstonia solanacearum, and Xanthomonas

campestris (Hao et al. 2012). Application of this new

finding could reduce energy consumption by 50 % or more.

The questions of major interest in this study were how this

new temperature regime may affect microbial community

in irrigation water and its practical implications. With the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11274-013-1583-y) contains supplementarymaterial, which is available to authorized users.

W. Hao (&) � C. X. Hong

Hampton Roads Agricultural Research and Extension Center,

Virginia Tech, Virginia Beach, VA 23455, USA

e-mail: weih913@vt.edu

123

World J Microbiol Biotechnol

DOI 10.1007/s11274-013-1583-y

current protocol, the majority of microorganisms in water

are killed at 95 �C, leaving a ‘‘biological vacuum’’ which is

prone to invasion by plant pathogens through other avenues

such as contaminated plants and planting materials

(Schumann and D’Arcy 2006). Heat treatment at 48 �C

does not compromise the entire microbial community in

irrigation water since this temperature is lower than the

lethal temperature for many microorganisms (Baker 1962;

Schumann 1991). The specific objectives of this study were

to (1) characterize the bacterial community structures in

heat-treated irrigation water and compare with those at

25 �C (control), and (2) explore the potential applications

of this shift in bacterial community composition.

Materials and methods

Water samples

Water samples were taken from a 0.8-ha reservoir at a local

nursery in eastern Virginia. This reservoir was replenished

with rain and runoff water from overhead irrigation of an 8-ha

ornamental plant production area. Three 1-l surface water

samples were collected in sterile bottles from the center of the

reservoir in February and May 2012. Water temperature and

other water quality parameters were recorded on-site using a

DS5X multiprobe Datasonde (Hach�, Loveland, CO, USA)

(Table S1). Water samples were then transported to the lab and

immediately used for heat treatment experiments.

Heat treatments

Water samples were placed at 25 �C (as control) and heat-

treated at 42 and 48 �C for 48 h. The heat treatment of 42 �C

was also evaluated because it effectively controlled several

Phytophthora species in water (Hao et al. 2012). For each

treatment, three 1-l replicates of water samples were trans-

ferred to Erlenmeyer flasks and placed in incubators (Percival

Scientific, Inc., Perry, IA, USA) at designated temperatures.

The incubators were calibrated in advance and temperature

was monitored during the experiment using a HOBO Pen-

dant� temp/light data logger (Onset Computer Corporation,

Bourne, MA, USA). Once the temperature treatments were

completed, water was passed through 10-lm nylon membrane

filters to remove larger debris and algae then analyzed using

both culture-dependent and independent strategies.

Culture-dependent strategy

Plating

One hundred microliters of the filtrate was evenly spread

on Nutrient Agar (NA) (DifcoTM, Detroit, MI, USA) in a

10-cm diameter Petri dish using a sterile glass spreader.

Three NA medium plates were used for each replicate

sample and incubated at 28 �C for 2 days.

Colony counts, PCR and SSCP analysis

Emerging colonies were categorized into nine groups by

color, texture, and size. Colony counts of individual groups

were recorded. About 20–30 % of individual colony

groups were subcultured from each treatment into 48-well

plates containing NA medium, and incubated at 28 �C for

2 days. So there were in total of 140–150 colonies were

selected from each treatment.

Colony PCRs were performed with primers 16S f968

and r1401 to amplify the hypervariable regions V6–V8 of

16S rDNA (Nubel et al. 1996; Peixoto et al. 2002).

Reagents supplied with Takara Taq (Takara Bio Inc.,

Japan) were used in 25 ll reactions containing 2.5 ll

109 PCR buffer (containing 1.5 mM MgCl2 in final mix-

ture), 0.2 mM of each dNTP mixture, 0.4 lM of each

primer, 18.4 ll DNA-free nanopure water, and 0.5 unit of

Takara Taq. The bacterial colonies were picked directly

from the culture plates and added to the PCR mixture.

Cycling conditions included an initial denaturation at

96 �C for 2 min, followed by 40 cycles of denaturation at

94 �C for 0.5 min, annealing at 55 �C for 0.5 min, and

extension at 72 �C for 1 min. For the last cycle, the

extension time was increased to 10 min.

Single-strand conformation polymorphism (SSCP)

analyses were performed for the colony PCR products as

described by Kong et al. (2003) with minor modifications.

In brief, the amplicons were mixed with loading dye

(1:4, v/v) and denatured by heating at 96 �C for 10 min

then chilled on ice. Two microliters of each PCR product

was electrophoresed in 8 % polyacrylamide gels (acryl-

amide/bisacrylamide = 29:1) at 220 V for 2.5 h in chilled

19 TBE buffer. After electrophoresis, the gels were stained

with silver nitrate (Beidler et al. 1982). SSCP banding

patterns of individual colonies were defined with the aid of

a 100 bp DNA ladder (Promega Corp., Madison, WI,

USA). Colony PCR products were grouped by SSCP fin-

gerprint and selected for DNA sequencing. All PCR pro-

ducts were sequenced for SSCP banding patterns which

had no more than ten. For the other banding patterns,

25–30 % of PCR products were selected for DNA

sequencing.

Culture-independent strategy

Genomic DNA extraction

The remaining filtrate (999.7 ml) was concentrated through

a combination of centrifugation and 0.2-lm filtration. In

World J Microbiol Biotechnol

123

brief, the filtrate was centrifuged at 12,2009g for 50 min,

pellets were saved, and supernatants were passed through

0.2-lm filters. The UltraClean Microbial DNA Isolation

Kit (MO BIO Laboratories, Inc, Carlsbad, CA, USA) was

used to extract genomic DNA from the pellets following

the instruction manual. The PowerWater DNA Isolation

Kit (MO BIO Laboratories, Inc, Carlsbad, CA, USA) was

used to extract genomic DNA from the 0.2-lm filters. Total

genomic DNA from both pellets and filters were combined

then used for DGGE analysis.

PCR amplification and DGGE

For DGGE analysis, 16S rRNA fragments containing three

hypervariable regions V6–V8 were amplified with primers

GC-clamp-EUB f933 and EUB r1387 (Kawai et al. 2002).

The PCR mixture (50 ll) was adapted to contain 5 ll

109 PCR buffer (containing 1.5 mM MgCl2 in final mix-

ture), 0.03 nM BSA, 0.2 mM of each dNTP mixture, 0.4 lM

of each primer, 19.3 ll DNA-free nanopure water, 1 unit of

Takara Taq, and 10 ll DNA extract. A hot-start PCR was

performed at 94 �C for 4 min, followed by touchdown PCR

with the following parameters: the denaturation was set at

94 �C for 1 min, annealing temperature was initially set at

65 �C and then decreased by 1 �C every cycle (1.5 min)

until it reached 55 �C. Twenty additional cycles were car-

ried out with the same annealing temperature at 55 �C.

Extension was carried out at 72 �C for 2.5 min. PCR com-

pleted with a final extension step for 7 min at 72 �C.

DGGE was performed using a Bio-Rad DcodeTM Uni-

versal Mutation Detection System (Bio-Rad, Hercules, CA,

USA) according to the manufacturer’s instructions. PCR

products were electrophoresed in 1-mm thick 8 % poly-

acrylamide gel (acrylamide:bisacrylamide = 37.5:1) in

19 TAE buffer. Gels were prepared with a denaturant

concentration of 40–60 %. Electrophoresis at 60 �C was

run at 70 V for 16 h. After electrophoresis, the gels were

stained with silver nitrate (Beidler et al. 1982).

DGGE gels were digitally documented using the Epi

Chemi II darkroom and analyzed using LabWorks software

(UVP Laboratory, Upland, CA, USA). Each lane was

detected automatically with the width and length adjusted

manually. Bands in each lane were detected automatically

and corrected manually.

DNA reisolation and cloning

Unique bands from each temperature treatment were

selected from the DGGE gels. DNAs were re-isolated from

the acrylamide gel using a ‘‘crush and soak’’ method with

minor modifications (Sambrook and Russell 2001). In

brief, bands were excised using sterile gel cutting tips

(BioExpress, Kaysville, UT, USA), transferred to 1.7-ml

tubes and washed briefly with 0.5 ml TE buffer. The gel

fragment was then crushed using a sterile pestle in 400 ll

elution buffer (0.5 M NH4OAc, 10 mM EDTA), incubated

at 37 �C overnight in an incubation shaker to allow DNA

diffusion from the gel then centrifuged at 11,7509g for

5 min to pellet the acrylamide. The supernatant was

transferred and DNA precipitated in 1 ml 100 % ethanol

with 16 ll 5 M NaCl at -20 �C overnight, then centri-

fuged at 11,7509g for 20 min. The pellets were vacuum

dried and re-suspended in 20 ll TE buffer. The extracted

DNAs were re-amplified with 16S rRNA primers EUB

f933 and EUB r1387 (Kawai et al. 2002) using the same

cycling conditions as colony PCR. These PCR amplicons

were cloned using the pGEM-T Easy Vector System

(Promega Corp., Madison, WI, USA). Randomly picked

white (positive) colonies were PCR amplified with the

plasmid primers T7 (50-TAA TAC GAC TCA CTA TAG

GG-30) and SP6 (50-ATT TAG GTG ACA CTA TAG AA-30)under the same cycling conditions as 16S rRNA colony PCR.

PCR products were grouped by SSCP banding patterns and

representatives from each group were selected for

sequencing.

Sequence analysis

All selected PCR products were sequenced at the

Advanced Genetic Technologies Center, University of

Kentucky. Sequences were edited using the BioEdit

Sequence Alignment Editor v. 7.1.3.0 (Hall 1999), and

subjected to BLAST search (http://blast.ncbi.nlm.nih.gov/

Blast.cgi) to identify the closest matches in GenBank.

Consensus phylogenetic trees for the partial bacterial 16S

rDNA sequences were built using the maximum likelihood

(Holmberg et al. 2009) based on the Tamura-Nei model

(Tamura and Nei 1993) in MEGA 5 (Tamura et al. 2011).

Reference sequences were collected from previous fresh-

water studies and reviewed by Newton et al. (2011). Nearly

full-length sequences of 16 rRNA ([1,300 bp), which were

the best available sequences, were used.

Data analysis

Culturable bacteria recovery from each temperature treat-

ment was summarized by taxonomic class based on the

colony PCR–SSCP and DNA sequence analysis data.

Subsequently, the relative abundance of five classes of

bacteria recovered was calculated by heat treatment for

each sampling date. For each of the two most abundant

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123

bacterial classes: Bacilli and c-Proteobacteria, their

recovery was further tallied by subgroup and accordingly

the relative abundance of individual subgroups was com-

puted and compared among the three temperature treat-

ments by sampling date.

For bacteria detected via culture-independent strategy,

non-metric multidimensional scaling (NMDS) analysis was

used to investigate the degree of similarity of bacterial

communities among temperature treatments. A binary

matrix of the DGGE band patterns was created based on

the presence (1) or absence (0) of bands which was then

used to generate a visual figure of distance by using the

Euclidean distance measure. NMDS analysis was carried

out using the PAST statistical software (version 2.17c)

(Hammer et al. 2001). For each NMDS, a stress value was

reported to indicate the degree of correspondence between

obtained and forecasts ranks (Clarke and Ainsworth 1993).

Results

A total of 280 partial 16S rRNA sequences from subcul-

tures and 118 sequences from DGGE have been deposited

in the GenBank under accession numbers JX870915 to

JX871216, JX628692 to JX628749, and JX657293 to

JX657330 (Table S2). Four phyla of bacteria were detected

in this study with Proteobacteria and Firmicutes being the

most abundant (Figs. 1, 2). The vast majority of the Fir-

micutes were detected by the culture-dependent strategy

from subcultures, while Bacteroidetes were detected

exclusively and Actinobacteria mostly by the culture-

independent strategy via DGGE (Fig. 2). All the Firmicutes

detected were Bacilli. The Proteobacteria detected were in

three major classes: a-Proteobacteria, b-Proteobacteria,

and c-Proteobacteria. Majority of the a-Proteobacteria

were detected by the culture-independent strategy. Like-

wise, more c-Proteobacteria and b-Proteobacteria were

detected via DGGE.

Comparing culturable bacteria among temperature

treatments

There was a stark contrast in the bacterial community

composition between heat-treated water and the control

(Figs. 1, 2). Overall, Bacilli accounted for about 50 % of

the total subcultures. Bacilli in water heat treated at 48 �C

were at least twice as abundant as the control for both the

February and May 2012 samples (Fig. 1a). A similar

increase in the Bacilli abundance also was observed at

42 �C in the February but not in May 2012 samples. As

illustrated in Fig. 1b, the subgroups accounting for this

increased abundance at 42 and 48 �C were Bacillus clusters

A and D, Paenibacillus, and Brevibacillus. Comparatively,

c-Proteobacteria accounting for more than 20 % of the

total subculutres became more abundant in water at 42 �C

but not at 48 �C when compared to the control (Fig. 1a).

The vast majority of this increased abundance was from

Pseudomonadaceae subgroup (Fig. 1b).

Comparing bacteria from culture-independent strategy

among temperature treatments

There also was a clear shift in the bacterial community

composition detected via culture-independent strategy

between heat-treated water and the control (Figs 2, 3). The

three temperature treatments differed by the DGGE band

position and their relative intensity at each sampling date

(Fig. 3). The dissimilarity of the DGGE patterns among the

three treatments is clearly shown by their distances as

estimated by a fair goodness of fit (stress value of Feb

samples: 0.16, stress value of May samples: 0.18) in the

NMDS analysis.

The phylogenetic analysis of the 16S rRNA gene

sequences of clones obtained from excised DGGE bands

also revealed numerous changes in bacterial community

composition among the three treatments (Table 1; Fig. 2).

Bacilli were only detected at 48 �C and they all were in the

cluster E. Actinobacteria were only found at 25 �C. In

c-Proteobacteria (Fig. 2b), the lineage gamV (Xanthomo-

nadaceae) contained clone sequences from both the 42 and

48 �C treated water, and the lineage gamIV (Pseudomo-

nadaceae) only contained sequences from the 42 �C treat-

ment. The cluster F had sequences from the control.

Sequences in a cluster G were originated from 42 �C. In

a-Proteobacteria (Fig. 2b), except the cluster H, all the

other clone sequences were from heat-treated water at both

42 and 48 �C. In b-Proteobacteria (Fig. 2b), the sequences

in the lineage betII (Burkholderiaceae) were from the

control. In Actinobacteria (Fig. 2a), all the clones, includ-

ing those in the lineage acI (Actinomycetales), were orig-

inated from the control. In Bacteroidetes (Fig. 2a), the

lineage bacI (Chitinophagaceae) was mostly at 42 and

48 �C, all other sequences were from both the control and

heat-treated water.

Discussion

This study demonstrated a bacterial community compo-

sition shift in irrigation water treated at 42 and 48 �C

as compared to the control at 25 �C. Culturable bacteria

in the genera Bacillus, Paenibacillus, and Brevibacillus

World J Microbiol Biotechnol

123

dominated the 48 �C-treated irrigation water, and cultur-

able bacteria in the family Pseudomonadaceae increased

in abundance at the 42 �C-treated irrigation water. The

DGGE-detected Bacillus also originated in irrigation

water treated at 48 �C while those Pseudomonadaceae

mostly originated from 42 �C. Likewise, bacteria in sev-

eral clusters of a-Proteobacteria and Xanthomonadaceae

of c-Proteobacteria detected via DGGE also originated

from water at 42 and 48 �C. These findings may have

practical implications.

0

10

20

30

40

50

25°C 42°C 48°C 25°C 42°C 48°C

Feb-12 May-12

Rel

ativ

e ab

unda

nce

of c

ultu

rabl

e ba

cter

ia (

%)

Sample date and temperature treatment

Actinobacteria -Proteobacteria

-Proteobacteria -ProteobacteriaBacilli

(a)

0 10 20 30 40

Others

Brevibacillus

Paenibacillaceae

Bacillaceae others

Bacillaceae Cluster D

Bacillaceae Cluster C

Bacillaceae Cluster B

Bacillaceae Cluster A

0 10 20 30 40

25°C 42°C 48°C

0 20 40 60 80 100

-Proteobacteria Cluster F

Enterobacteriales(gamII)

Pseudomonadaceae(gamIV)

0 20 40 60 80 100

(b)Bacilli

-Proteobacteria

Relative abundance of culturable bacteria (%)

Feb 2012 May 2012

Fig. 1 a Relative abundance of

culturable bacteria detected in

irrigation water samples in

February and May 2012 after

48 h temperature treatments at

25, 42 and 48 �C; b relative

abundance of culturable

bacterial families/clusters

within each of Bacilli and c-

Proteobacteria among three

temperature treatments

World J Microbiol Biotechnol

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The increased abundance of Bacillus and Paenibacillus

species in the 48 �C-treated water, and that of Pseudomo-

nas species in the 42 �C-treated water was not unexpected.

Warth (1978) indicated that the optimum temperatures of

mesophilic Bacillus species are between 30 and 45 �C, but

some thermophiles are able to grow as high as 76 �C. The

optimal growth temperature of Paenibacillus species is

28–42 �C, and the maximum growth temperature lies

between 50 and 55 �C (Bosshard et al. 2002; Scheldeman

et al. 2004). The heat tolerance of those bacteria, in the

family Bacillaceae and Paenibacillaceae, is related to their

ability to differentiate into endospores. When heat kills the

vegetative cells, the heat-resistant spores can survive and

germinate when environmental conditions improve (Wil-

son et al. 1992; Hahn et al. 2004). The optimum temper-

atures for growth of Pseudomonas species are variable.

However, some species, such as P. aeruginosa, can grow at

temperatures as high as 42 �C (Tsuji et al. 1982).

Increased abundance of Bacillus, Paenibacillus, and

Pseudomonas species could be indicative more antagonis-

tic activities against plant pathogens in heat-treated water.

A large number of Bacillus, Paenibacillus, and Pseudo-

monas species are known to have antimicrobial activities

against various plant pathogens. For instance, Bacillus

amyloliquefaciens RC-2, B. cereus L7, B. megaterium KL

39, B. pumilus INR7, B. subtilis strains QST713 and GB03

have been used as biocontrol agents for different plant

diseases, including gray mold, damping-off, and powdery

mildews (Raupach and Kloepper 1998; Paulitz and Bel-

anger 2001; Yoshida et al. 2001; Jung and Kim 2003;

Romero et al. 2004; Santoyo et al. 2012; Zhao et al. 2012).

The underlying mechanisms include competition, parasit-

ism, antibiosis, and induced systemic resistance (ISR).

Some Paenibacillus species, for example, P. polymyxa

strains and P. koreensis, produce antibacterial (Piuri et al.

1998; Martin et al. 2003; Shaheen et al. 2011) or antifungal

compounds (Singh et al. 1999; Chung et al. 2000; Beatty

and Jensen 2002; Tupinamba et al. 2008; Deng et al. 2011).

Pseudomonas species, such as P. aeruginosa, P. fluores-

cens, P. putida, are the most extensively utilized for bio-

logical control of many seedling diseases and root rots on

various crops, due to their ability to scavenge available

ions in soil, produce antibiotics, and trigger ISR (Duijff

et al. 1993; Yang et al. 1994; Punja 1997; Maier and

Soberon-Chavez 2000; Paulitz and Belanger 2001; Viji

et al. 2003; Loper et al. 2007; Loper et al. 2008). The

increased abundance of these groups in heat treated water

likely enhances the biological control activities against

plant pathogens in this aquatic environment.

The a-Proteobacteria and Xanthomonadaceae of c-Pro-

teobacteria detected via DGGE were mainly from heat-

treated water in this study. The identity of these a-Prote-

obacteria cannot be determined at this time, because the

closest matches in GenBank have not been identified to

order/family level. Some a-Proteobacteria species have

been reported to be heat-tolerant. For instance, Rhodo-

spirillum centenum, a thermo-tolerant a-proteobacterium

producing heat-resistant cysts under certain nutritional

conditions, grows from 39 to 47 �C, and its survival cysts

can stand for 55–75 �C (Stadtwald-Demchick et al. 1990).

Rhodomicrobium vannielii also forms the exospore-like

cysts with a moderate heat resistance (Imhoff 2006). Very

few studies reported the survival temperatures of Xantho-

monadaceae. Our results showed some a-Proteobacteria

and Xanthomonadaceae are heat resistant up to 48 �C in

water, however, the practical implications of these bacteria

in heat-treated water are yet to be determined.

Lack of Actinobacteria detection from heat treated water

was expected. Antinobacteria were observed only at 25 �C

regardless of detection method (Fig. 1a). No endospores or

microcysts have been found for Actinobacteria. Hahn et al.

(2003) reported that freshwater representative Actinobac-

teria were very small (\0.1 lm3) in size with thin cell

walls. These characteristics of Actinobacteria may be

responsible for their susceptibility to extreme environ-

mental factors, such as, heat stress.

In conclusion, the new heat treatment resulted in an

increased abundance of Bacillus, Paenibacillus, and

Pseudomonas species in irrigation water. Some members

in these groups are known to be antagonistic against

numerous plant pathogens; thus this new heat treatment

may enhance the biological control activities in treated

water, providing a hidden advantage in promoting sus-

tainable disease management. Further investigations are

warranted to test this hypothesis and elucidate the eco-

logical functions of those a-Proteobacteria and Xantho-

monadaceae detected in heat-treated irrigation water via

DGGE.

bFig. 2 Phylogenetic analyses of partial 16S rDNA sequences of

bacteria detected from irrigation water samples collected in February

and May 2012 after 48 h temperature treatments at 25 �C (triangle), 42

(circle) and 48 �C (diamond) via cultures-dependent (filled) and

-independent strategies (unfilled). The frequency of each sequence

detected is listed in parentheses. The maximum likelihood method

based on the Tamura–Nei model of 1,000 replicates was used.

Bootstrap values higher than 50 are shown with lineage and clades

designated from Newton et al. (2011). a Actinobacteria, Bacilli, and

Bacteroidetes with Escherichia coli (Z83205) and Erwinia tasmanien-

sis (NR_074869) as outgroup; b a-Proteobacteria, b-Proteobacteria,

and c-Proteobacteria with Methanotorris formicicus (NR_028646) and

Thermococcus acidaminovorans (AB055120) as outgroup

World J Microbiol Biotechnol

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Fig. 2 continued

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Fig. 3 DGGE fingerprints and

non-metric multidimensional

scaling (NMDS) analysis of

DGGE banding patterns of

bacterial 16S rRNA fragments

in irrigation water collected in

a February and b May 2012

after 48 h temperature

treatments at 25 �C (unfilled

triangle), 42 (filled circle), and

48 �C (gray diamond). The

DGGE was run at 70 V for 16 h

with a gel denaturant

concentration of 40–60 %. One

replicate out of three each

treatment was shown. Arrows

represent selected bands.

L represents 100 bp DNA

ladder. The Eculidean model

was used as the similarity

measure for NMDS

World J Microbiol Biotechnol

123

Acknowledgments This work was supported by a grant from the

USDA National Institute of Food and Agriculture—Specialty Crop

Research Initiative (Agreement #: 2010-51181-21140). We would

like to thank Drs. Boris Vinatzer, Anton Baudoin, Erik Stromberg,

Michael Benson, Giovanni Cafa, and Ping Kong for their valuable

advice during this study, and we also would like to thank Patricia

Richardson for assisting with water sampling and proofreading this

manuscript.

References

Baker KF (1962) Principles of heat treatment of soil and planting

material. J Aust Inst Agric Sci 28(2):118–126

Baker KF, Matkin OA (1978) Dectection and control of pathogens in

water. Ornamentals Northwest April–May:12–13

Beatty PH, Jensen SE (2002) Paenibacillus polymyxa produces

fusaricidin-type antifungal antibiotics active against Leptosp-

haeria maculans, the causative agent of blackleg disease of

canola. Can J Microbiol 48(2):159–169. doi:10.1139/w02-002

Beidler JL, Hilliard PR, Rill RL (1982) Ultrasensitive staining of

nucleic acids with silver. Anal Biochem 126:374–380

Bosshard PP, Zbinden R, Altwegg M (2002) Paenibacillus turicensis

sp. nov., a novel bacterium harbouring heterogeneities between

16S rRNA genes. Int J Syst Evol Microbiol 52:2241–2249.

doi:10.1099/ijs.0.02105-0

Cayanan DF, Dixon M, Zheng YB, Llewellyn J (2009) Response of

container-grown nursery plants to chlorine used to disinfest

irrigation water. HortScience 44(1):164–167

Chung YR, Kim CH, Hwang I, Chun J (2000) Paenibacillus koreensis

sp. nov., a new species that produces an iturin-like antifungal

compound. Int J Syst Evol Microbiol 50:1495–1500

Clarke KR, Ainsworth M (1993) A method of linking multivariate

community structure to environmental variables. Mar Ecol Prog

Ser 92:204–219

Deng Y, Lu ZX, Lu FX, Wang Y, Bie XM (2011) Study on an

antimicrobial protein produced by Paenibacillus polymyxa JSa-9

isolated from soil. World J Microbiol Biotechnol 27(8):1803–

1807. doi:10.1007/s11274-010-0638-6

Duijff BJ, Meijer JW, Bakker PAHM, Schippers B (1993) Sidero-

phore-mediated competition for iron and induced resistance in

the suppression of Fusarium wilt of carnation by fluorescent

Pseudomonas spp. Neth J Plant Path 99(5–6):277–289. doi:10.

1007/bf01974309

Geldreich EE (1996) Pathogenic agents in freshwater resources.

Hydrological Process 10(2):315–333

Hahn MW, Lunsdorf H, Wu QL, Schauer M, Hofle MG, Boenigk J,

Stadler P (2003) Isolation of novel ultramicrobacteria classified

as Actinobacteria from five freshwater habitats in Europe and

Asia. Appl Environ Microbiol 69(3):1442–1451. doi:10.1128/

aem.69.3.1442- 1451.2003

Hahn MW, Stadler P, Wu QL, Pockl M (2004) The filtration-

acclimatization method for isolation of an important fraction of

the not readily cultivable bacteria. J Microbiol Methods

57(3):379–390. doi:10.1016/j.mimet.2004.02.004

Hall TA (1999) BioEdit: a user-friendly biological sequence align-

ment editor and analysis program for Windows 95/98/NT.

Nucleic Acids Symp Ser 41:95–98

Hammer O, Harper DAT, Ryan PD (2001) PAST: paleontological

statistics software package for education and data analysis.

Palaeontologia Electronica 4(1):1–9

Hao W, Ahonsi MO, Vinatzer BA, Hong CX (2012) Inactivation of

Phytophthora and bacterial species in water by a potential

energy-saving heat treatment. Eur J Plant Pathol 134(2):357–

365. doi:10.1007/s10658-012-9994-4

Holmberg AIJ, Melin P, Levenfors JP, Sundh I (2009) Development

and evaluation of SCAR markers for a Pseudomonas brassic-

acearum strain used in biological control of snow mould. Biol

Control 48(2):181–187. doi:10.1016/j.biocontrol.2008.10.016

Hong CX, Moorman GW (2005) Plant pathogens in irrigation water:

challenges and opportunities. Crit Rev Plant Sci 24(3):189–208.

doi:10.1080/07352680591005838

Imhoff JF (2006) The phototrophic alpha-proteobacteria. Prokaryotes:

a handbook on the biology of bacteria, Vol 5, Third Edition:

Proteobacteria: alpha and beta subclasses. Springer, New York.

doi:10.1007/0-387-30745-1_2

Jung H-K, Kim S-D (2003) Purification and characterization of an

antifungal antibiotic from Bacillus megaterium KL 39, a

biocontrol agent of red-papper phytophthora blight disease.

Korean J Microbiol Biotechnol 31(3):235–241

Kawai M, Matsutera E, Kanda H, Yamaguchi N, Tani K, Nasu M

(2002) 16S ribosomal DNA-based analysis of bacterial diversity

in purified water used in pharmaceutical manufacturing pro-

cesses by PCR and denaturing gradient gel electrophoresis. Appl

Environ Microbiol 68(2):699–704

Kong P, Hong CX, Richardson PA, Gallegly ME (2003) Single-

strand-conformation polymorphism of ribosomal DNA for rapid

species differentiation in genus Phytophthora. Fungal Genet Biol

39(3):238–249. doi:10.1016/s1087-1845(03)00052-5

Loper JE, Kobayashi DY, Paulsen IT (2007) The genomic sequence

of Pseudomonas fluorescens Pf-5: insights into biological

control. Phytopathology 97(2):233–238. doi:10.1094/phyto-97-

2-0233

Loper JE, Henkels MD, Shaffer BT, Valeriote FA, Gross H (2008)

Isolation and identification of rhizoxin analogs from Pseudomonasfluorescens Pf-5 by using a genomic mining strategy. Appl

Environ Microbiol 74(10):3085–3093. doi:10.1128/aem.02848-07

Maier RM, Soberon-Chavez G (2000) Pseudomonas aeruginosa

rhamnolipids: biosynthesis and potential applications. Appl

Microbiol Biotechnol 54(5):625–633

Martin NI, Hu HJ, Moake MM, Churey JJ, Whittal R, Worobo RW,

Vederas JC (2003) Isolation, structural characterization, and

properties of mattacin (Polymyxin M), a cyclic peptide antibiotic

produced by Paenibacillus kobensis M. J Biol Chem

278(15):13124–13132. doi:10.1074/jbc.M212364200

McPherson GM, Harriman MR, Pattison D (1995) The potential for

spread of root diseases in recirculating hydroponic systems and

their control with disinfection. Meded Fac Landbouwkd Toegep

Biol Wet Univ Gent 60(28):371–379

Newton RJ, Jones SE, Eiler A, McMahon KD, Bertilsson S (2011) A

guide to the natural history of freshwater lake bacteria. Microbiol

Mol Biol Rev 75:14–49. doi:10.1128/MMBR.00028-10

Table 1 Summary of bacterial presence in irrigation water at dif-

ferent temperatures collected in February and May 2012, identified by

partial 16S rDNA sequences of excised DGGE bands

Taxonomy February 2012 May 2012

Class 25 �C 42 �C 48 �C 25 �C 42 �C 48 �C

Actinobacteria ? – – ? – –

Bacilli – – ? – – ?

Bacteroidetes ? ? ? – ? ?

a-Proteobacteria ? ? ? ? ? –

b-Proteobacteria ? ? – – – –

c-Proteobacteria – ? – ? ? ?

? Indicating the presence of the bacterial class

World J Microbiol Biotechnol

123

Nubel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI,

Ludwig W, Backhaus H (1996) Sequence heterogeneities of

genes encoding 16S rRNAs in Paenibacillus polymyxa detected

by temperature gradient gel electrophoresis. J Bacteriol

178(19):5636–5643

Paulitz TC, Belanger RR (2001) Biological control in greenhouse

system. Annu Rev Phytopathol 39:103–133

Peixoto RS, Coutinho HLD, Rumjanek NG, Macrae A, Rosado AS

(2002) Use of rpo B and 16S rRNA genes to analyse bacterial

diversity of a tropical soil using PCR and DGGE. Lett Appl

Microbiol 35(4):316–320

Piuri M, Sanchez-Rivas C, Ruzal SM (1998) A novel antimicrobial

activity of a Paenibacillus polymyxa strain isolated from

regional fermented sausages. Lett Appl Microbiol 27(1):9–13

Punja ZK (1997) Comparative efficacy of bacteria, fungi, and yeasts

as biological control agents for diseases of vegetable crops. Can

J Plant Pathol 19(3):315–323

Raupach GS, Kloepper JW (1998) Mixtures of plant growth-

promoting rhizobacteria enhance biological control of multiple

cucumber pathogens. Phytopathology 88:1158–1164

Romero D, Perez-Garcia A, Rivera ME, Cazorla FM, de Vicente A

(2004) Isolation and evaluation of antagonistic bacteria towards

the cucurbit powdery mildew fungus Podosphaera fusca. Appl

Microbiol Biotechnol 64(2):263–269

Runia WT (1995) A review of possibilities for disinfection of

recirculation water from soilless cultures. Acta Horticulturae

382:221–228

Runia WT, Vanos EA, Bollen GJ (1988) Disinfection of drainwater

from soilless cultures by heat-treatment. Neth J Agric Sci 36(3):

231–238

Sambrook J, Russell DW (2001) Molecular cloning: a laboratory

manual, 3rd edn. Cold Spring Laboratory Press, Cold Spring

Harbor, NY

Santoyo G, del Carmen Orozco-Mosqueda M, Govindappa M (2012)

Mechanisms of biocontrol and plant growth-promoting activity

in soil bacterial species of Bacillus and Pseudomonas: a review.

Biocontrol Sci Technol 22(8):855–872. doi:10.1080/09583157.

2012.694413

Scheldeman P, Goossens K, Rodriguez-Diaz M, Pil A, Goris J, Herman

L, De Vos P, Logan NA, Heyndrickx M (2004) Paenibacillus

lactis sp. nov., isolated from raw and heat-treated milk. Int J Syst

Evol Microbiol 54:885–891. doi:10.1099/ijs.0.02822-0

Schumann GL (1991) Plant diseases: their biology and social impact.

APS Press, St. Paul, MN

Schumann GL, D’Arcy CJ (2006) Essential plant pathology, 1st edn.

APS Press, St. Paul, MN

Shaheen M, Li JR, Ross AC, Vederas JC, Jensen SE (2011)

Paenibacillus polymyxa PKB1 produces variants of polymyxin

B-type antibiotics. Chem Biol 18(12):1640–1648. doi:10.1016/j.

chembiol.2011.09.017

Singh PP, Shin YC, Park CS, Chung YR (1999) Biological control of

fusarium wilt of cucumber by chitinolytic bacteria. Phytopathol-

ogy 89(1):92–99. doi:10.1094/phyto.1999.89.1.92

Stadtwald-Demchick R, Turner FR, Gest H (1990) Physiological

properties of the thermotolerant photosynthetic bacterium,

Rhodospirillum centenum. FEMS Microbiol Lett 67(1–2):139–

143. doi:10.1016/0378-1097(90)90183-q

Stanghellini ME, Rasmussen SL (1994) Hydroponics—a solution for

zoosporic pathogens. Plant Dis 78(12):1129–1138

Tamura K, Nei M (1993) Estimation of the number of nucleotide

substitutions in the control region of mitochondrial-DNA in

humans and chimpanzees. Mol Biol Evol 10(3):512–526

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S

(2011) MEGA5: molecular evolutionary genetics analysis using

maximum likelihood, evolutionary distance, and maximum

parsimony methods. Mol Biol Evol 28:2731–2739

Thomson SV, Allen RM (1974) Occurrence of Phytophthora species

and other potential plant pathogens in recycled irrigation water.

Plant Dis Rep 58:945–949

Toze S (1999) PCR and the detection of microbial pathogens in water

and wastewater. Water Res 33(17):3545–3556

Toze S (2006) Water reuse and health risks—real vs. perceived.

Desalination 187(1–3):41–51. doi:10.1016/j.desal.2005.04.066

Tsuji A, Kaneko Y, Takahashi K, Ogawa M, Goto S (1982) The effect

of temperature and pH on the growth of eight enteric and nine

glucose non-fermenting species of gram-negative rods. Micro-

biol Immunol 26(1):15–24

Tupinamba G, da Silva AJR, Alviano CS, Souto-Padron T, Seldin L,

Alviano DS (2008) Antimicrobial activity of Paenibacillus

polymyxa SCE2 against some mycotoxin-producing fungi.

J Appl Microbiol 105(4):1044–1053. doi:10.1111/j.1365-2672.

2008.03844.x

van Os EA (1999) Closed soilless growing systems: a sustainable

solution for Dutch greenhouse horticulture. Water Sci Technol

39(5):105–112

Viji G, Uddin W, Romaine CP (2003) Suppression of gray leaf spot

(blast) of perennial ryegrass turf by Pseudomonas aeruginosa

from spent mushroom substrate. Biol Control 26(3):233–243.

doi:10.1016/s1049-9644(02)00170-6

Warth AD (1978) Relationship between heat-resistance of spores and

optimum and maximum growth temperatures of Bacillus species.

J Bacteriol 134(3):699–705

Wilson M, Epton HAS, Sigee DC (1992) Biological control of fire

blight of hawthore (Crataegus monogyna) with fluorescent

Pseudomonas spp under protected conditions. J Phytopathol

136(1):16–26. doi:10.1111/j.1439-0434.1992.tb01277.x

Yang C-H, Menge JA, Cooksey DA (1994) Mutations affecting

hyphal colonization and pyoverdine production in Pseudomo-

nads antagonistic toward Phytophthora parasitica. Appl Environ

Microbiol 60(2):473–481

Yoshida S, Hiradate S, Tsukamoto T, Hatakeda K, Shirata A (2001)

Antimicrobial activity of culture filtrate of Bacillus amylolique-

faciens RC-2 isolated from mulberry leaves. Phytopathology

91(2):181–187. doi:10.1094/phyto.2001.91.2.181

Zhao S, Pan WB, Ma C (2012) Stimulation and inhibition effects of

algae-lytic products from Bacillus cereus strain L7 on Anabaena

flos-aquae. J Appl Phycol 24(5):1015–1021. doi:10.1007/

s10811-011-9725-9

World J Microbiol Biotechnol

123