Rhizobacteria Underground Biocontrol
Microbiologist Robert Kremer and technician Lynn Stanley compare callus tissue
specimens growing in a 24-well tissue culture plate.
Roots, as they grow through the soil, ooze organic substances in their wake.
If they're scratched, roots exude even more of these substances, thus providing
a veritable feast for the plant's microbial friends and foes.
Among a crop plant's friendliest microbes are those that attack weeds.
Agricultural Research Service
microbiologist Robert J. Kremer at Columbia, Missouri, is rooting for the
underground weed foes called deleterious rhizobacteria (DRB.)
In general, rhizobacteria live on plant roots or reside in the rhizosphere,
a soil zone spanning a few millimeters around roots, where they feed on plant
juices. Deleterious rhizobacteria, which poison plants, don't invade roots as
parasites but may enter through wounds.
Once weakened by DRB, weeds are less able to compete with other plants for
soil nutrients, moisture, and sunlight. The weakened weeds also become more
vulnerable to other control measures.
DRB-produced toxins trigger plant cells to produce excessive hormones that
keep seeds from germinating, or they damage the plants by putting life
processes in overdrive, Kremer says.
Damage might show up as slow plant growth. Under a microscope, cell fluids
may appear discolored. Pressure from a fluid buildup may cause cell walls to
break and leak, replenishing the DRB diet.
Now Kremer says DRB may become the basis for a commercial bioherbicide
against one of the worst weeds in the West: leafy spurge, Euphorbia
esula. A Eurasian native, the weed now infests at least 29 states and
costs 4 of them--Montana, Wyoming, and North and South Dakota--an estimated
$144 million annually.
Unpalatable to cattle and horses, the weed, if left unattended, overruns
untilled land, degrading it even for wildlife habitat. On land suited only for
livestock grazing, controlling the weed with chemical herbicides alone may be
temporary at best and too expensive--to say nothing of being possibly harmful
to other plants.
Although Kremer is no friend of leafy spurge, he's interested in it
"mainly as a model plant for tissue-culture-based research on
rhizobacteria," he says. Kremer and his colleagues have developed a time-
and labor-saving procedure, using leafy spurge tissue culture, to identify
which bacteria to test on whole plants for their power to wage biological weed
warfare. It may just be the first weed cell culture used to evaluate the
potential of DRB.
Rhizobacteria that first piqued researchers' interest increased crop growth
either directly, by stimulating hormones, or indirectly, by producing
antibiotics that inhibited plant disease microbes. But in the early 1980s,
scientists discovered DRB that reduced seed germination and seedling growth of
sugar beets, wheat, and citrus.
That's about the time Kremer started looking for microbes that might combat
velvetleaf, cocklebur, jimsonweed, and other weeds in cultivated crops of the
Midwest and South. His quest also led him to DRB on leafy spurge.
A leafy spurge root with samples of media (calcium alginate and semolina) used
to inoculate test plant tissue with various DRB strains.
In the soil around velvetleaf seedling roots, Kremer found the most
prevalent microbes included pathogenic, or disease-causing, fungi in the genera
Fusarium and Alternaria. He dipped seeds in a liquid suspension
of the microbes and planted them for greenhouse tests.
"When the velvetleaf seedlings were 2 weeks old," he says,
"we found top growth reduced as much as 88 percent, compared with
Meanwhile, ARS entomologist Neal R. Spencer, then at Stoneville,
Mississippi, was researching the insect Niesthrea louisianica,
which feeds only on seeds of velvetleaf, prickly sida, and spurred anoda.
The pathogenic fungi hitchhike on the insect. Then, as the insect feeds on
not quite mature but viable weed seeds, the fungi infect the weakened remains.
In field tests, Kremer and Spencer found only about 5 percent of velvetleaf
seeds survived and germinated after the insect-fungi duo attacked.
Before bioherbicides for velvetleaf, leafy spurge, or any other weed will
merit commercial interest and farmers' acceptance, Kremer says, research must
address a number of issues. For example, low-cost methods must be developed to
produce effective microbial strains that remain viable in storage and
consistently work well under field conditions.
ARS, with its long-term perspectives and wide-ranging expertise, is
developing a knowledge base that will be needed to foster biological control
Teamwork by Kremer and Spencer begun in the 1980s recurred in the '90s when
Spencer, now at Sidney, Montana, began research on flea beetles that lay their
eggs at the base of leafy spurge plants. [See "Leafy Spurge Is Reunited
with an Old Enemy," Agricultural Research, April 1994, pp. 20-22.]
ARS plant pathologist Anthony J. Caesar, also at Sidney, discovered that
larvae feeding on roots spread not only DRB, but also pathogenic fungi
throughout leafy spurge's vast root systems. Caesar and Spencer are evaluating
the effectiveness of the insect/fungi interactions in a 5-year areawide
integrated pest management (IPM) research project.
To identify which DRB might best control leafy spurge, Kremer first applied
strains to seedling roots in growth chamber and greenhouse tests. Strains that
worked best could then be field tested. Later, as an alternative to growing
seedlings, he grew leafy spurge cells suspended in a liquid culture medium. He
then inoculated samples of the liquid suspensions with test bacteria, and after
48 hours, he treated the samples with dye.
Only leafy spurge cells killed by the bacteria were stained by the dye.
Selecting the most deadly bacteria by measuring color changes with a
spectrophotometer took less labor than measuring root lengths and provided more
consistent results, but the process was still tedious.
Kremer decided that testing the bacteria on callus tissue might be even more
efficient. Callus is a mass of plant cells not quite as fully developed as
cells in a whole plant. The technology for tissue culture had already been
developed in the mid-1980s by ARS plant physiologist David G. Davis at Fargo,
North Dakota. Callus samples were available from ARS chemist Gary D. Manners at
Further underpinning the idea was previous research completed in Kremer's
lab by Thouraya Souissi. She had found that the bacteria caused callus cells to
grow poorly, become discolored, and even to leak severely.
Souissi and Kremer developed a visual rating system to compare the toxic
powers of various DRBs using plates with 24 wells--sort of micro test
tubes--containing nutrients for the callus. Into each well they placed
half-gram pieces of leafy spurge callus and a drop of DRB. Forty-eight hours
later, the researchers recorded observations on each DRB in a database.
Microbiologist Robert Kremer displays healthy leafy spurge roots.
Testing the various rhizobacteria on multiwell tissue-culture plates
requires little laboratory space and takes only one-twentieth of the time
needed for whole-weed studies.
"We think this is a cost-effective way to speed assessment of superior
biological control agents so they can be put to practical use sooner,"
Development of "living pesticides" may also be speeded by changing
technologies for producing and storing formulations. [See "The Next
Bioherbicide?" Agricultural Research, June 1995, p. 20, and
"New Process Keeps Biocontrols Alive Longer," Agricultural
Research, October 1998, p. 22.]
Kremer says further advances in bioherbicide technology may depend on
insights gained through basic research. To take a closer look at some of the
most effective DRBs identified in routine screening, he used an electron
Strains of Pseudomonas fluorescens and Flavobacterium
balustinum seemed to paste themselves directly to the plant cells and
set up "factories" for producing toxic compounds. The tissue-cultured
cells--like infected cells in whole plants--absorbed the compounds and became
deformed and stunted.
Some 10 million cells of one strain of P. syringae applied to the
standard half gram of tissue culture reduced the weight of fresh callus by 20
percent within 48 hours. And in preliminary field tests, the isolate has shown
further promise, reducing leafy spurge root development. So far, the
researchers have found a dozen examples of North American rhizobacteria that
were highly toxic to the tissue and showed promise in field tests.
Yet to be screened are about a third of some 2,500 rhizobacteria cultures
isolated from weedy Euphorbia species of Europe, as well as North
America. Of those rhizobacteria screened already, about 30 percent are highly
toxic to leafy spurge. Some of these may prove better suited than others for
mass-production and commercial use.
Many DRB feed only on the juices of specific plants. But how do the
scientists know whether some DRBs are not going to harm economically useful
plants growing in the area where they might be applied?
"That's always a question we try to answer systematically through our
research," says Kremer. Rhizobacteria not native to an area where they
might be used merit greater scrutiny than native ones. Before outdoor tests can
be done, scientists will conduct extensive indoor studies.--By
Ben Hardin, Agricultural
Research Service Information Staff.
Robert J. Kremer is in the
System and Water Quality Research Unit, Room 302, ABNR Bldg., University of
Missouri, Columbia, MO 65211; phone (573) 882-6408, fax (573) 884-5070.
Neal R. Spencer and
Anthony J. Caesar are at
the USDA-ARS Northern Plains
Agricultural Research Laboratory, 1500 North Central Ave., Sidney, MT
59270; phone (406) 482-2020, fax (406) 482-5038.
"RHIZOBACTERIA Underground Biocontrol Allies?" was
published in the October 1998 issue of Agricultural Research magazine.
to see this issue's table of contents.