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Stapp. C. & G. Spicher 1951A. Untersuchugan uhar Mie WA-ufh,-•e_ 'ra --

im Boden [Research on the activity of 2,,4-D in the aoilJ7

Zentralblatt Mr Bakteriologie, Parasitenkundo, &c. 11. Abt. Bd. 1C8.

Heft 4/1, pages 113-126.

"- The stability of the selectiv4 hormonal herbicide, 2,4-D, in

the soil i± certainly one of its most important properties; for only

so long as the 2,,4-P acid maintains itself in the soil can the

desirod weed control be effected. The use of 2,4-D is advisable,

since it affords a weed-free environment for planting, but it can be

used repeatedly only with the assumption that, after the desired

period of control, the herbicide is inactivated or removed from ths

Ssoil. Bince the 2,4,D must below a certain concentration before""

planting, this problem deserves a lot of attention. A slight

over-abundance in the substrate can ruin the crop (Audus, 1949;

Bouillene-Walrand, 1952; Jensen & Petersen, 1952).

It is well known that most 2,4-D disappears from treated soil

within a few weeks of treatment. This disappearance is attributed to

microorganisms which metabolize 2,4-D. Using the mperfusion"

technique, Audus (1949) has plotted the 2,,-1D concentration against

time; after treatment, he distinguishes three phasea: (a) a slight

reduction of the detectible 2,4-D1, the result of ad~sption onto soil

colloids; (b) a phase during Ltich the 2,4-D level remains abaV' %he

same; (o) a rapid dIs earance of 2,4-.D. The curve is typica.

10-1-

• -

2of those reflecting biological procossos in the soil /j 11-17

and Audus attributes the "detoxication" to the work of MLLoroouu1.juan.

With repated treatment of the soil, tho second phase is hastened.

Autoclaving or addition of a bactericide inhibits the activity.

Im impure culture of soils innoculated with the active orgeniam

2,4-D was brosken down. in the ame way (Neuvan & Thomas, 1950);

Audus (1950:1951) finally isolated an organism which ijas able to survive

on synthetic media with 2,4-D as its only carbon-source. According to

his work, this bacterium is obviously a member of the Bacterium globi-

forme group of Lochead & Taylor. Tater Jensen & Petersen (1952) ider.-

tified t•wo more 2,1-D bacteria, a "Flavobacterium aquatile," and a

so-callod "Bacterium 2," which they stated bore some relationship to

the form de3cribed by Audus.

SIdentification of a 2,4-D Bacterium

Proceeding from the earlier work (Stapp & Preter, 1952; Stapp &

V:otter, 1953) :, also succeeded in isolating the active organism which

in pure culture on solid or liquid media was able to inactivate the

sodium salt of 2,4-D (when not otherwise specified, "2,4,-D" will here

signify the ecdi = salt thereof). Composition of the medium: Na2HPO4 -

=2o0 0.'^o1 4 3 0.1%, Ki 0.0o%, MsSO4 .7H20 0.02%, 2,s4-D 0.1%, Agar

2.0 to 0.1'.

Tha eczonatration of the possibility of fluid-media culture was

a signi-Mica-nt advance. This was possible by spreading the medium with

sufficicr.t surface-area for a good oxygen supply (as the work of

Akm•ine, 1951 and of St#app & Wetter 1953 indicate is required), and

above all an addition of 01% agar, which Audus (1949, 1951) had

M 7.

3had already indicated. It is also a good idea to permit the autoclavedculture flasks to stand at room temperature for a day before innoculation

so that the media an bbsorb oxygen.

The yield of the im;0u-e ulture of 2,"-D bacteria can be enhanced

by repeated moistening of Uhe sample with a 0,% solution of 2,4-D, with

a cress test following each moistening) This procedure can be repeated

until finally when a "small amount" of the "enriched Souil is Introduced

into the flask containing the 2,4-D-containing medium, there is an

immediate reduction in the 2,4-D concentration in the flask.

Puro culture at tiret was difficult, until it became evident that

the 2,4-D bacterium is very sensitive to temperature. The unusually

low upper limit of temperature tolerance was demonstrated thusly:

Per each temperature series p 2 cc of sterile medium was inocculated

with a young cr.ture, and this was aowod to remain lu minutes in

a constant-temperature water bath. The following different temperatures

were used: 25, 30, 35, 40, 45, 50, and 550 C. Solid 2,4-D medium was

then inncoulated from the temperature-treated aliquot, and the survival

of the bacterium noted. The test was replicated four times. In all

cases the bacteria survived temperatures of 4CPC or less. A ten-minute

exposure at 40 0C greatly reduced the bacteria, while at 450C the

tolerance limit was already exceeded.

Even in the most diverse soils, the addition of 2,L•-D led to a

similar increase in the 2,4-D bacteria. This was accompanied, in all

inpure cultures, by an increase in Bacillus meraterium.

The tat(4s of the "Mamial of Determinatige Bacteriology" by

0 Bergey, and the so-called w"kerman-keym (1949) or its Gormn translatiov

--- --- --- --- --.... ..........-- --. ,.----"-I., .-.- ... .. . L. .

by IteYn (1952) were used in the -Ictermination of genus to wvhich the

isolated 2,94-D bacterium belongs, Both indicate the gemas Flavobacterium.

Specific determination was-considerably more difficult * The key of the

"Manual of Determinative Bacteriology" takes us to three different

species: F. actuatil..F breve, and Z* soae

All throe are non-motile, do not dilute gelatine, or do so only very

slowly, leave litims unchanged,, and do not reduce nitrate to nitrite.

(tiry in their tempeature optima are Visy clearly differentiable.

From a farther investigation of propertiesoit was inferred that

our 2,,4-D bacterium is closest to Flavobacterium aqail, insofar as

the Inference covld be dram =n the basis of the published descriptions

of properties. We contrast the two species below:

The colony of F. aquatile has a ye~llow-brown center and a colorless

0 margin; our bacterium has a puzre ypflow colony.

F.aqatile dilutes gelatine only very slowly, our bacterium not

at all.

F.aquatile occurs as singjles,, pairs,, and chains; ou bacterium

occurs as singles or pairsL, not as chains.

F. aqatl tolerates but does not. require o37~gen; our bacterium

requires oztygen.

Above afll, they differ in size (or length-width ratio): when F. aczuatile

is 0.5 micra wide it is oout 2.5 lolag; wehen our bacterium is that wide,,

it is only about 0.8 micra long.

The 2,,4,D bacterium identified by Jensen & Petersen (1952) as

F, aquatile fits the description of the "Maival of Determinative Bact-

O eriology" in size (larger than ours). Curiously, the bactetlum of

_A4

SJensen & Peterseon does not grow on a semisolid agar-medium nor an earth-U extract, but devolole well on a glucose medium* Dar bacterium, an the

other hand, does very wvll on nutrient solution plus 0.1% agar, and is

markedly held back when 1% glucose is added.

In an attempt to deter!A4 n ;ww our 2,4-D bacterium compares with

strains which have been determined by other authorities as Flavobacterium

aquatile in a series of tests four of these strains were compared with

our bacterium (Table 3). All five strains are very s9-ilar, but display

a few distinctions, these having to do with chain-formation, colonr-

color, ability to thin telatine, and gowth in nutrient bouillon.

Strain 1 is closely related to strain 2, and strain 3 to strain 35. Or

* 2,,4-D bacterium is intermediate, with a special form of colony in aga

and in gelatine.

0In an examination of properties not specified in the Xsnualf,

more extensive differences became evident, when neither acid nor gas

formed in the bouillon-carbohydrate media (Table 4). Here (in Table

4) our bacterium is again. intermediate, but closer to strain 3 and 35.

Thi, descrease or retardation of its developeont in the presence of

glucose and lactose is especially remarkable. 04Ily our 2,,4-D bacteriua

persists on the 2,4-D medium; even after a week's incubation, none of

the others develops an adaptation to 2,4-D.

Audus (1949) hypothecated that the long first phase In the break-

down of 2,4-D in previously untreated soil was the time during which the

norm&all inactive bacteria a1apt themelves to 2,4,-D metabolism.

Jensen & Petersen (1952) considered it unlikely that an organism with an

ensyme system capable of this break-dowu would be conn in a previously

U___________,__________

untreated 0oil. Thy considered the? 2#". bacteriam to be & Favo-

bacterium aquatil. adapted for the bmak-4mi of cyclic ompomLde.

Sinme, ho"ever none of the Investiated strains of Fiavobacterium

aquatile from untreated soils has ever 8ucceeded in utlising 2,jv-D, we

consider it apPop-iate to look upon our 2,4-D bacterium as new, and

propose for it the nme nlavobaoteriuR - new species.

The Break-doim of 2jiv-D by Flavobacterium W

It seemed to us very Important to tompare the activity of pure

and imp•re culture of Flavobacterium porgrinum, to see whether the

imjpure culture may eventually contain still other organisms capable

of 2,j-D break-dom. The possibility arises, that if the 2,4-.D

bacterium can be induced to break down 2,4-D in fluid medium, the

portion of the break-dom attributable to the 2,,-D bacterium (Strain

(j D) can easily be ascertained.

The nutrient described cc page 11w ma doubled in concentration,

but with the same amount of earth-sitract, and 20 00 placed in eaoh of

a number of 10-cc flasks$ and autoolaved. One group wa ionoculated

with Strain D, the other with a small aumimt of a soil sample

enriched with the 2,4-D bacteriu.

In pure vilture the principal phase of 20,-" breok-down usually

starts the fBst day, and after 2.5 d the bacteria have inactivated

half of the 2,4-D supplied. In Impire cultures on the other hand, the

principal phase begins on the thirdl day, and the half-rmy phint is

reached between the fourth and fifth days. Therefore llavobacterium

vsremrinua is solely responsible for the nutrient-break-down, and in

this respect Bacterium meateorium, which is usually found with it, in

insignificant.

LWIF .o, that the d~isappearance of 294,D in various soil&

proceeds at different rates. However, the results of research on thehave

reason for these differences, up until now, & been equivocal.

EThero follows a short review of literature: Erie,, 1947; Aka e,

1951; Stapp & Wetter, 1953; Kitchell & MArth, 1946; Hnks, 1947; rom'

& Mitchell, 1948; Krone & Hamner, 1947-1

With the aid of fluid culture it can be shomn that *oils of

various origins contain a factor which, depending on the origin of the

#oil, differentially affects the rate of growth of the 2,4-D bacterium.

The finely sifteds oilwere placed in twice their weight of

distilled water, were heated 30 minutes in the autoclave, centrifuged,

and filtered through a mbrane filter (No. 10 of the membrane filter

company in Gottingen). A part of the resulting extract was added to a

esidlar amount of the doubly-concentrated nutrient solution, and 20 co'

placed in a mimber of 100-cc. flasks, sterilized, innoculated ,i"th

Flavobacterium , and incubated at 259C.

The addition of soil extract speeds up the breakdow (see fig.

"3a. and 3b.); if the tims is propitious, the tim required to break

down half the 2,$4 is reduced from 72 to &0 hours. %ae most stronl

active soils were, in c - experience, humic to very.humic fine sandy

loam, with no carbonates, probably alluvial, and used as madows. The

The results of several other workers have been taken to indicate that

the rate of 2,4-D inactivation is dependent on the sum of several factors

whose total effecto determines the effective activity. In our experience,

one of thee" factors, as already pointed out by Audus (1952) is a water-

soluble pwv -stimiiant %ich occurs in wing amounts. Our factor0

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