Washington, D.C. California Canada

1150 18 th St., NW, Suite 1000 712 Fifth St., Suite A 275 Slater St., Suite 900

Washington, D.C. 20036 Davis, CA 95616 Ottawa, Ontario K1P 5H9

Phone: (202) 223

‐ 4392 Phone: (530) 757

‐ 1245 Phone: (613) 247

‐ 6285 Technology Sciences Group Inc.

1150 18 th Street, N.W.

Suite 1000

Washington, D.C. 20036

Direct: (202) 828-8956

Fax: (202) 872-0745

E-Mail: bmilesont@TSGUSA.com

Beth E. Mileson, Ph.D., DABT

Senior Managing Toxicologist

Becker Underwood, Inc.

801 Dayton Avenue

Ames, IA 50010

June 6, 2012

Identification, Distribution and Natural Populations of Bacillus pumilus

Bacillus pumilus strain BU F

‐ 33 is a new active ingredient proposed by Becker

‐ Underwood for

use in agriculture for preventative control and suppression of plant root pathogens (e.g.,

Rhizoctonia spp

. and Fusarium spp .) and/or to increase the amount of nodulation by nitrogen

‐ fixing bacteria by legumes. Bacillus pumilus is a Gram

‐ positive endospore

‐ forming bacterium

that is common in the environment around the world. B. pumilus bacteria are found in all types

of soil, and have been identified in a variety of coastal and aquatic environments as well. The

use of this proposed new active ingredient is not expected to increase the levels of B. pumilus in

the environment because B. pumilus is ubiquitous in nature and has been identified and

isolated from samples of a variety of media including, soil, water, aquatic invertebrates and

growing plants.

B. pumilus bacteria are ubiquitous in soil in the United States (US) and around the world, and

occur naturally in all types of agricultural soil. The wide distribution of B. pumilus in a variety of

soil types is reflected in the examples from the literature presented here. B. pumilus was

identified and isolated from rhizosphere samples collected from field

‐ grown spring wheat,

winter wheat, and barley (Juhnke et al., 1987). These rhizosphere samples were collected on

an agronomy farm near Bozeman, Montana where the soil type is described as Bozeman silt

loam. In a separate study, B. pumilus was isolated from soil samples collected in the San

Joaquin Valley of California (Halverson, 2000). The soil type in the San Joaquin Valley where

these samples of B. pumilus were collected is categorized as saline

‐ sodic. Samples of soil

collected in an agricultural area of the Netherlands revealed that B. pumilus was naturally

occurring in permanent grassland, short

‐ term arable land, and long

‐ term arable land (Garbeva

et al., 2003). The results of this study indicate that B. pumilus bacteria are naturally present in

agricultural soil whether the soil has been planted recently or not. Soil samples collected inTurkey h

xylanase

collecte d

2004). T

soil. Th e

around t

B. pumil u

coworke

Sea, and

seawate r

(Merina e

positive r

predomi

pumilus

Hole, M a

The artic

the amo u

characte

prevalen

to be in t

Bacillus p

level in s

Copies o f

for addit

Sincerel y

Beth E. M ave been u s

(Yasinok et

d from volca

he cacti coll

e identificati o

he world is a

u s has been

rs (1999) sa m

the Coral S e

r itself. B. p

e t al., 2011)

r ole in the n

n ant bacter i

h as also be e

a ssachusett s

les cited ab o

u nt of the m

ristics for id e

t bacteria i n

t he range of

p umilus stra

oil.

f the cited r e

ional infor m

y

,

M ileson s ed as a sou r

al., 2010). B

n ic areas of

ected from t

o n of B. pu m

a n indicator

identified i n

m pled wate

e a of the Pa c

p umilus is al s

. Sea grass i

utrition of m

i a in this be n

e n identified

s (Oguntoyi n

o ve that ide n

m icrobe in th

e ntification

n each samp l

10 6 to 10 7 c

in BU F

‐ 33 a

e ferences a r

m ation. r ce of B. pu m

B

. pumilus w

the central

t he volcani c

m ilus in soil a

of the ubiq u

n aquatic en v

r and marin e

c ific Ocean a

s o a natural

i s often coa t

m arine plan t

n eficial biofi l

in water sa

n bo, 2007).

n tified B. p u

e environm e

purposes. T

l e in the pa p

c olony formi

ccording to

r e provided i m ilus that w a

w as identifie d

Baja Califor n

area were g

a nd rhizosp h

u ity and ab u

v ironments

e invertebr a

a nd found B

.

component

t ed with a b i

s, and B. pu m

l m under na

mples colle c

u milus in en v

e nt, but inst

T he fact tha

p ers cited su

ng units (cf u

the label di r

i n the attac h B.

a s isolated a

d in rhizopl a

n ia Sur mou n

g rowing in w

h eric sample

u ndance of t

as well as i n

a tes in the S e

. pumilus in

of the biofi

i ofilm of ba c

milus has b e

a tural condit

c ted in the A

v ironmental

ead focuse d

t B. pumilu s

ggests that

u ) per gram

r ections wo u

h ment. Ple a pumilus Na t

B e

a nd used to p

a ne samples

ntain range

w eathered r o

e s througho u

his microbe

n the soil. Iv a

e a of Japan,

sea sponge s

lm that for m

c teria that p

e en identifi e

t ions (Merin a

A tlantic Oce a

samples di d

d on the mo l

s was amon g

population l

of soil. App

u ld not incr e

a se feel free t ural Distrib

e cker Under w

Page 2

p roduce

of cacti

(Puente et a

o ck rather t h

u t the US an d

.

a nova and h

the Okhots k

s and in the

m s on sea gr a

lay a signifi c

e d as the

a et al., 201 1

a n at Wood s

d not deter m

l ecular

g the most

l evels are li k

lication of

e ase the nat

to contact m ution

w ood

2 of 3

a l.,

h an

d

h er

k

a ss

c ant

1 ). B.

s

m ine

k ely

ural

m eB. pumilus Natural Distribution

Becker Underwood

Page 3 of 3

References

1. Garbeva P, van Veen JA, van Elsas JD. 2003. Predominant Bacillus spp. in agricultural soil

under different management regimes detected via PCR

‐ DGGE. Microbial Ecology.

45:302

‐ 316.

2. Halverson LJ, Jones TM, Firestone MK. 2000. Release of intracellular solutes by four soil

bacteria exposed to dilution stress. Soil Sci. Soc. 64; 1630

‐ 1637.

3. Ivanova EP, Vysotskii MV, Svetashev VI, Nedashkovskaya OI, Gorshkova NM, Mikhailov

VV, Yumoto N, Shigeri Y, Taguchi T, Yoshikawa S. 1999. Characterization of Bacillus

strains of marine origin. International Microbiol. 2; 267

‐ 271.

4. Juhnke ME, Mathre DE, Sands DC. 1987. Identification and characterization of

rhizosphere

‐ competent bacteria of wheat. Applied Environmental Microbiology. 53(12);

2793

‐ 2799.

5. Merina M, Lipton AP, Wesley G. 2011. Isolation, characterization and growth response

of biofilm forming bacteria Bacillus pumilus from sea grass, Halodule pinifolia off

Kanyakumari. Indian Journal of Marine Sciences. 40(3); 443

‐ 448.

6. Oguntoyinbo F A, 2007. Monitoring of marine Bacillus diversity among the bacteria

community of sea water. African Journal of Biotechnology. 6 (2); 163

‐ 166.

7. Puente ME, Bashan Y, Li CY, Lebsky VK. 2004. Microbial populations and activities in the

rhizoplane of rock

‐ weathering desert plants. I root colonization and weathering of

igneous rocks. Plant Biol. 6 (2004) 629

‐ 642.

8. Yasinok AE, Biran S, Kocabas A, Bakir U, 2010. Xylanase from a soil isolate, Bacillus

pumilus : gene isolation enzyme production, purification, characterization and one

‐ step

separation by aqueous

‐ two

‐ phase system. World J. Microbiol. Biotechlol. 26; 1641

‐

1652.