Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol84.Issue1-2.Apr2000:

Scientia Horticulturae 84 (2000) 179±189

The use of genetically engineered bacteria to control
frost on strawberries and potatoes. Whatever
happened to all of that research?
R.M. Skirvina,*, E. Kohlerb, H. Steinerc, D. Ayersc,
A. Laughnanc, M.A. Nortond, M. Warmunde
a

University of Illinois, Department of Natural Resources and Environmental Sciences,
258 ERML, 1201 W. Gregory Dr., Urbana, IL 61801, USA
b
University of Illinois, Department of Natural Resources and Environmental Sciences,
307 ERML, 1201 W. Gregory Dr., Urbana, IL 61801, USA
c
University of Illinois, Department of Natural Resources and Environmental Sciences,
258 ERML, 1201 W. Gregory Dr., Urbana, IL 61801, USA
d
University of Illinois, Department of Natural Resources and Environmental Sciences, 258 ERML,
1201 W. Gregory Dr., Urbana, IL 61801, USA
e

University of Missouri, Department of Horticulture, 1-87 Agriculture Building,
Columbia, MO 65211, USA
Accepted 28 July 1999

Abstract
The identi®cation of biological ice nucleating agents and their importance in frost induction and
prevention is discussed. The discussion also includes information about the researchers who did the
work, their original investigations, and struggles with government agencies to introduce their
products. The original research was initiated independently by a group of atmospheric scientists in
Wyoming and a group of plant pathologists in Wisconsin. They both discovered that ice does not
form randomly but is initiated on a nucleating site which is associated with particular bacterial
species, especially Pseudomonas syringae. From this original discovery has come commercial
products that are used to prevent frost (FrostbanTM [the generic name for bacteria that lack the
genes coding for the ability to form ice crystals on the leaves of crop plants (Oei, H.L., 1999. Genes
and Politics: The Recombinant DNA Debate. Chatelaine Press, Burke, VA)] and Blightban1),
manufacture snow (Snomax1), reduce the incidence of ®re blight (Blightban1), and as an aid for
food concentration and texturing. The moral and ethical questions encountered by the scientists

*

Corresponding author. Tel.: ‡1-217-333-1530; fax: ‡1-217-333-4777.

0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 9 7 - 7

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performing the original research helped to establish the rules by which biotechnology research is
carried out today. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Pseudomonas syringae; Pseudomonas ¯uorescens; Xanthomonas campestris; Snomax;
Blightban; Frostban; Ice nucleation; Frost

1. Introduction
Prior to 1967 the existence of biological ice nucleation and its effects on living
organisms at subfreezing temperatures was unsuspected (Upper and Vali, 1995).
It was well known that water could be supercooled from ÿ38C to ÿ58C in natural
samples, while distilled water samples could be supercooled from ÿ128C to
ÿ208C, but the source of ice nucleation activity remained speculative. The topic

was particularly important to plant scientists because of frost injury (Siminovitch
and Scarth, 1938) and to atmospheric scientists who studied hail and snow. Hail, a
common phenomenon that causes widespread damage to crops and property, was
believed to form on a nucleus of inorganic material such as atmospheric dust
particles. The source of the dust was conjectural but was believed to be derived
from storms which lifted soil particles into the atmosphere, volcanoes, or from
meteoric material (Upper and Vali, 1995).
To determine the nucleating agent for snow and hail formation, Vali, an
atmospheric scientist (Upper and Vali, 1995) recalls collecting clean rain and
snow in clean plastic bags. The samples were analyzed to identify nucleating
sites. After a long winter of collecting and analyzing clean snow samples, in
desperation for one more sample, he took a sample of dirty snow from beneath his
daughter's swing set (Upper and Vali, 1995). The dirty snow caused ice
nucleation at a higher temperature than any other sample tested. The nucleus of
the snow was found to contain organic matter. About 1970 Vali and Russell
Schnell teamed up to study the phenomenon. Schnell showed that decaying
grasses and leaves were the likely source of ice nuclei in humus. The freezing
nuclei from decaying leaves were called leaf-derived nuclei (LDN). In
conjunction with Leroy Maki's laboratory, they eventually found that the agent
was a bacterium, Pseudomonas syringae (Maki et al., 1974).

About the same time the cloud physics research was going in Wyoming,
scientists at the University of Wisconsin were studying the mechanism whereby
some corn plants were cold injured following inoculation with Helminthosporium
turcicum (now known as Setosphaeria turcica) (northern corn leaf blight). Hoppe
et al. (1964) observed that corn lines treated with ground up infected corn leaves
showed considerable frost damage; control plants showed little damage. They
®rst hypothesized that the leaf blight fungus made their plants sensitive to frost.
Later, however, they found that leaves treated with healthy ground up leaves were

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181

just as likely to have frost damage as those treated with infected leaves. Thus,
frost damage was due to an agent other than leaf blight (Upper et al., 1972). In
1973 Stephen Lindow joined the group and was assigned the task to ®nd the agent
responsible for frost damage. About this time they noted that plant extracts that
had been sitting around in solution for a day or two caused more frost damage
than fresh extracts. These observations suggested that a bacterium was involved.
Arny et al. (1976) isolated and identi®ed strains of P. syringae that were

responsible for inducing ice nucleation.
P. syringae,which is not pathogenic to humans, produces a protein complex in
its outer membrane that can serve as a nucleus for ice crystal formation. In a
population of bacteria, those having ice nucleating active (INA‡) sites frequently
constituted 1% or less of the total bacterial population present; however, the
values can vary by time of the year and location (Hirano and Upper, 1995). The
protein complex initiates ice crystal formation at temperatures slightly below
freezing, killing or damaging plants at temperatures they otherwise could
withstand. Bacteria then become established in frost-damaged plant tissue where
they acquire nutrients as they are released by injured cells (Hirano and Upper,
1995). According to Chen et al. (1995) there are two general types of ice
formation in biological systems: homogeneous and heterogeneous. In homogeneous ice nucleation, nuclei form spontaneously in liquid (usually below
ÿ388C). In heterogeneous ice nucleation, nucleation is induced by external
factors such as bacteria, fungi, plants and insects (as reported by Klassen, 1986).
Thus, pure water without nucleating sites to serve as catalysts can be supercooled
to around ÿ408C (Brown, 1997) without freezing. Water in plant tissues can be
supercooled to ÿ58C, without harming the plants, yet when the ice nucleating
bacteria (INA‡) are present, ice formation can begin at about ÿ18C (Klassen,
1986).
An immediate application became apparent: if the protein complex responsible

for the ice crystal formation could be removed, it might be possible to delay frost
formation, and thus extend the growing season, as well as reduce or eliminate the
need for expensive frost protection systems such as irrigation and gas heaters.
Losses due to frost damage have been estimated at about 1.5 billion dollars per
year (Jaroff, 1986). Reducing the incidence of frost and the amount of damage it
causes would increase both crop yields and quality.
Both Schnell and Lindow quickly began to investigate ways to eliminate the
protein complex responsible for frost formation. Working independently, they
took two different approaches. Schnell's approach was to eliminate the bacteria
from plant surfaces. To do this, he chose to isolate a virus that would kill only the
ice nucleating bacteria, thus preventing the production of the protein complex. To
help fund his work, Schnell was licensed by the University Genetics Co. of
Norwalk, Connecticut. Frost Technology Corporation was set up to help
commercialize the results. Unfortunately, his approach had its shortcoming: the

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virus had anti-bacterial activity only at the time of application and for a short time

thereafter. Since bacterial populations can increase rapidly and viral application
was a single event, the virus was effective only temporarily.
Lindow's approach, however, appeared to show more promise. Lindow, now at
the University of California at Berkeley, proposed to use recombinant DNA
technology to modify the bacteria to eliminate the protein that promotes freezing
(Baertlein et al., 1992). In other words, the bacteria would be genetically altered
not to carry the genetic instructions needed to produce the ice nucleating protein.
Furthermore, he proposed that when this new bacteria (INAÿ) was sprayed onto
plants at very high concentrations, naturally occurring bacteria (INA‡) would not
be able to compete. Unlike the method using virus, the new bacteria would be
effective for a longer period, reducing the necessity for exact timing of the
application. With this type of frost protection, it was estimated that strawberries,
for example, might survive temperatures as low as ÿ78C (Lindemann and Suslow,
1987). Advance Genetic Sciences (AGS) of CA marked the new bacteria as
FrostbanTM.
In September, 1983, Lindow obtained approval by the National Institutes of
Health (NIH) to test the bacteria on outdoor experimental potato plots in
Tulelake, northern California (Hall, 1987). This decision was met with
controversy and opposition (Maranto, 1986). On 12 April, 1984, the ®rst of
many legal battles was led by Jeremy Rifkin of the Foundation on Economic

Trends (Maranto, 1986). Rifkin sought to bar the NIH's decision on the basis that
they approved the project without an examination of the possible environmental
risks. Rifkin compared the modi®ed bacteria to``foreign organisms introduced
into the USA like the Japanese beetle and the gypsy moth,'' both of which have
been found to be harmful to plants (Jaroff, 1986). He also claimed that the
genetically engineered bacteria might multiply and change environmental
conditions. Since the protein could play an important role in the formation of
ice crystals that evolve into snow¯akes and raindrops, he maintained that
widespread use of the bacteria could alter rainfall patterns and distribution.
Advocates of the test countered that this was ridiculous because most rains begin
in the upper atmosphere where temperatures are so cold that ice crystals form
without the need for nucleating particles (Rhein, 1985). Nonetheless, the
opposition remained forceful and determined, and a temporary injunction against
the University of California Agriculture Experiment Station was ordered to begin
on 25 May, 1984.
AGS ®led an appeal immediately and later in 1984 asked for permission to test
the bacteria on strawberries in Monterey County, CA. In November, 1984, the
Environmental Protection Agency (EPA) gave permission for the ®elds to be
sprayed with Frostban, but again the tests were postponed by the county board
and residents. The delay was related to publicity concerning an earlier EPAapproved test that AGS had performed outdoors on fruit trees. The EPA had given

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183

permission for the test to be performed in a sealed greenhouse. The trees actually
had been in a greenhouse, but they had grown so that they no longer ®t in the
company's tallest greenhouse. AGS claimed that the experiment had been
contained because the trees had been injected with a syringe beneath their bark,
and no bacteria were exposed to the atmosphere. The EPA considered this to be in
violation of their testing rules, but later agreed that the injected trees showed no
sign of disease or death due to the bacteria. This event was bad publicity for the
company and their testing program (Sun, 1986a). As a result, when the proposed
test on strawberries was approved, the residents of Monterey County protested,
claiming AGS had failed to educate them about the testing and its possible
effects. AGS admitted to this and a 45-day ban on testing was put into effect in
February, 1985, and then again in March (Jaroff, 1986). During this period, many
Monterey citizens expressed concern at Jeremy Rifkin's claim that rainfall
patterns might be altered if the bacteria were released into the atmosphere (Jukes,
1987).

AGS turned their attention to other possible test sites in Contra Costa County in
California. They took all measures to educate the public in the area before testing,
and again they were thwarted, this time by an environmentalist group from
Berkeley called the Berkeley Greens (Jukes, 1987). On 24 April, 1985, the day
that the testing was to take place, the fence surrounding the test site was cut and
2200 of the 2400 strawberry plants to be tested were uprooted (Jukes, 1987).
On 13 May, 1985, the EPA ®nally granted Lindow permission to test the
bacteria on potatoes over a 3-year period in Tulelake, CA. Previously, many
potato farmers in the state had been in favor of the testing, but after all the bad
publicity, 460 people signed a petition to delay the test (Sun, 1986b; Hall, 1987).
Due to negative community feelings, the University of California temporarily
withdrew its support of Lindow and the experiment. Despite statements from the
EPA and NIH that the experiment was harmless, Lindow was forced to wait for
general approval. Lindow was quoted as saying ``[developing this test] was the
stupidest thing I've ever done. I wouldn't recommend it to anyone else Ð not
until people are more educated'' (Sun, 1986c). Finally the university approved the
test after 2 years of waiting. On 23 April, 1987, a Sacramento Superior Court
judge denied Rifkin's ®nal effort to stop the AGS experiment (Hall, 1987). This
was the ®rst such test to clear all state and federal regulatory hurdles and
withstand all legal challenges. The decision ended Rifkin's 4-year battle to block

these ®eld tests. The dif®culties and frustrations associated with these tests have
been scathingly summarized by Miller (1997).
AGS wasted no time, and the following day they sprayed a small test plot of
strawberries with Frostban in Brentwood, a small farming community 50 miles
east of San Francisco. 5 days later, Lindow began a similar test on potatoes at the
University of California Agricultural Experimental Station in Tulelake, CA.
Although the tests had court approval, protestors from``Earth First''and the``East

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Bay Green Alliance'' marched with signs, and vandals uprooted the plants and
poured salt and chlorine on the soil.
The AGS report to the 1985 conference on Biotechnology in Plant and Animal
Agriculture stated that the engineered form of the bacteria is``exactly identical to
the natural one except that it cannot cause nucleation of ice crystals'' (Adams,
1985). Once the gene for ice nucleation (inaZ) had been identi®ed, it was a
relatively simple job to clone the gene and introduce it into other organisms. The
identi®cation, use, and procedures used to engineer genes have been reviewed by
Panopoulos (1995) and Wolber et al. (1995).
1.1. Frostban1
In 1987 AGS and a small biotechnology company called DNA Plant
Technology merged to form a company called DNAP (pronounced dee-nap). In
1989, after 2 years of being on the back burner due to public opposition, DNAP
renewed interest in Frostban. They decided against using genetically altered
bacteria and concentrated on selecting naturally occurring bacterial strains
instead. This choice was made because registering a genetically engineered
product for a crop was so much more dif®cult and time consuming than
registering one that occurs naturally. The downside is that it could be dif®cult to
®nd the exact strain that is effective on a given plant. However, AGS isolated a
natural nonnucleating bacterial strain that was very similar to the genetically
engineered strain. Both strains were tested and proven to protect against frost
down to a temperature as low as 228F.
One of the scientists involved in the DNAP/AGS product testing studies was
Dr. Trevor V. Suslow (Extension Specialist, Department of Vegetable Crops,
University of California at Davis). He provided the following story of what
happened to the product called Frostban. Eventually four formulations of
Frostban were registered with the EPA. These included a three strain mixture and
one registration for each component strain (one strain of P. syringae and two
strains of P. ¯uorescens; one of these was A506). Small quantities (less than
100 kg) of Frostban A (the mixture) were sold commercially in states other than
California. This was necessary to complete the establishment of the trademark
name ``Frostban''. Frostban is a trademarked product name that is not strain
speci®c and would include naturally occurring and genetically engineered agents.
The development rights to Frostban were sold to Frost Technologies (Frost
Technology Corporation, 1992), which dropped their license after 1 year.
1.2. Blightban1 (Plant Health Technologies, Boise, ID)
Plant Health Technologies picked up the license for Frostban but according to
Suslow (personal communication) the company decided to ``. . . shy away from

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185

frost protection as a product market. They decided to focus on ®re blight control
and as a means to get into the biologicals `game' and develop the production and
distribution infrastructure [for] a more contained market where the perceived
value (disease control) was higher. They had to register Frostban A506 in CA and
add ®reblight control to the product label claims for federal and California.'' The
product was a type of P. ¯uorescens (A506) federally registered as a biological
pesticide marketed as Blightban A5061 (Lindow et al., 1996). The name
Blightban A5061 was selected because it better re¯ected the company's
marketing strategy and it was less likely to be confused with a chemical product
called Frostgard1 (Personal communication, Steve Kelley, Plant Health
Technologies). Blightban A5061 was demonstrated to have biological activity
for control of frost damage, fruit russetting, and ®re blight disease (Erwinia
amylovora) (Lindow et al., 1996). The nonvirulent P. ¯uorescens is reported to
overgrow and invade normal infection sites for Erwinia, so the ability to cause
infection is severely reduced (Lindow, 1987; Lindow et al., 1996).

2. Other uses for the technology
2.1. Snomax1 Snow inducer (York International)
About the time the court battles began, AGS discovered that the naturally
occurring bacterial nucleating protein complex, which triggered ice formation at
temperatures near freezing, could be used as an aid for making snow at ski
resorts. AGS, in conjunction with BioFrost (Hamilton, Ontario), developed a
powdered form of freeze-dried bacteria called Snomax that, when added to water
in snow making equipment, initiated freezing at higher temperatures than
conventional machines.
To make arti®cial snow, water normally is supercooled with compressed air
(the expensive part of snow-making) to about ÿ108C before it will crystallize.
The mechanics of snowmaking have been reviewed (Hoffman, 1998). With
Snomax ice can form at about ÿ38C (Snomax Technologies, 1999). The result is
a better quality product; old fashioned snow machines made pellets but Snomax
makes ¯akes that more closely resemble natural snow (Miller, 1995). This
ef®ciency (about 80%) has saved Canadian ski resorts at least $100 million per
year in energy costs (Kopvillem, 1985). However, the price of the Snomax
product can be so high that some people only use it with energy intensive snow
machines. AGS licensed the Snomax technology to Eastman Kodak, which began
marketing it to ski resorts in the fall of 1987. As of the spring of 1990, twelve
downhill and one cross country ski areas in Vermont, as well as a number of other
ski areas across the country, were using Snomax (Daley, 1990a,b). A speci®c area
is the Killington Ski area in Vermont (Hoffman, 1998), where 580 million gallons

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of water are used for snow making each year. They used 180 000±184 000 gallons
of water just to produce a foot of snow on one acre (Daley, 1990a). Killington
resurfaces their trails several times throughout the season due to loss of snow
from skier traf®c, steepness of trails, grooming operations, and weather.
Killington's snow-making capabilities would be severely limited without Snomax
says Carl Spangler, Vice President in charge of planning (Daley, 1990a). They
also have been able to `seed' clouds above ski areas to induce snow and control
weather (Daley, 1990b).
Eastman Kodak reported another application of Snomax. They have adopted
the technology to make a low energy cooling system at its Rochester, New York,
headquarters (Skerrett, 1993). Beginning in late November, when air temperatures are below freezing, a water/Snomax mix is sprayed through hundreds of
nozzles attached to towers standing in a 6 foot deep pond roughly the size of an
ice rink. This produces a very thick blanket of snow that gradually turns to ice.
The cold water beneath the ice is pumped to a heat exchanger where it chills the
water used for refrigeration. This project began in November of 1991, and since
has reduced peak electrical load by 90% from 389 kW to just 25 kW. Says project
engineer Kaj Huld, ``instead of using electricity or mechanical cooling, the cold
weather does all the work'' (Skerrett, 1993).
The use of Snomax has become so popular for making arti®cial snow that some
environmentalists claim that some ski resorts are making so much arti®cial snow
that rivers and streams are being drained at the expense of wildlife (Dellios, 1995;
Hoffman, 1998). Some individuals also express concern that the addition of
genetically altered bacteria to snow and the environment may be a health risk.
The Snomax people counter that the organism (P. syringae) used to make Snomax
occurs naturally and is prepared by freeze drying and grinding to yield a protein,
not bacteria, as the end product. The resulting pellets are then sterilized prior to
sale using ``the same type of equipment used to routinely sterilize surgical
instruments.'' The number of live organisms released is so small [according to
York International advertising] that ``. . .if Snomax were used at all of the
country's 70 ski resorts with snowmaking, the total release of live microorganisms would be no more that what could be recovered from 100 leaves in a
farmer's ®eld'' (Snomax Technologies, 1999).
2.2. Food processing
The food industry has also made use of bacterial ice nucleation. Watanabe and
Arai (1995) report that many food storage application involve the use of bacteria
for freeze-concentration. The use of freezing to remove water avoids loss of
volatiles and chemical deteriorations associated with heating. Because Pseudomonas strains are potentially pathogenic to humans, nonpathogenic strains of
Xanthomonas have been permitted for food use. Such a strain (X. campestris) has

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187

been isolated from tea (Watanabe and Arai, 1995). To avoid bacterial
contamination during concentrations, bacterial cells are contained in cellophane
or calcium alginate but still they can nucleate water at ÿ58C. The containment
device can be placed at any level in a solution and freezing will begin at that
point. This system has been used to concentrate egg whites, lemon juice, milk,
and strawberry jam. The strawberry jam was reported to be superior to that
produced using traditional heating for brightness, red color, and ¯avor. The
bacteria can also enhance the rate of freeze drying for such dif®cult to freeze
products as soy sauce and soybean paste. The uniform freezing observed with
bacteria has also facilitated better texturing of proteins for making meat
substitutes.
DNAP's selection and marketing of a nongenetically engineered organism for
frost control, was an interesting overall strategy to bypass the complex rules
associated with introduction of genetically engineered organisms. Other
researchers working in this area also may prefer to market products selected
from wild populations rather than with genetic engineering (Fall and Wolber,
1995). Later, after the waters of regulation and consumer acceptance of biotech
food products have been tested, genetic engineering companies may again choose
to introduce products developed using genetic engineering.
For biotechnology products to be economically successful they must positively
affect the consumer and provide real improvements in the quality of their life or
products to affect their lives such as better food ¯avor, texture and quality.
Snomax, for example, has been very successful across North America because it
provides the consumer higher quality snow for skiing. It is a tangible bene®t that
af¯uent consumers (skiers) can readily understand and one they are willing to pay
for. On the other hand, Frostban and its various applications lack obvious direct
consumer bene®ts; so their acceptance has been slower. For instance, the decision
to rename one of Frostban's components, P. ¯uorescens A506, as Blightban A506
was made to capture a market that was perceived to be larger than that for frost,
®re blight control.
Although genetically engineered organisms offer great potential bene®ts for
agriculture, the lack of immediate consumer bene®ts and the public's general fear
of new technologies has resulted in a general public reluctance to purchase or
accept these products, especially in Europe. Time overcame the environmental
opposition to the Frostban work. Ultimately it will be consumer acceptance and
demand that will make or break this emerging industry.
In conclusion, the basic research initiated by Gabor Vali, Steven Lindow and
Russell Schnell in the 1970s to study the role of bacteria in ice formation has
resulted in methods to control frost, produce snow for skiers, reduce the incidence
of ®re blight infection, as well as produce higher quality concentrated food
products. The development of these commercial products was controversial in
their time. However, the controversy forced researchers, environmentalists, and

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politicians to debate the scienti®c and moral importance of biotechnology. For
better or for worse, the research that was ®rst planned to protect strawberries and
potatoes from frost has resulted in unexpected industries, products, and a dynamic
view of biotechnology and its importance for plant and animal improvement.

Acknowledgements
This report was supported in part by funds provided by the University of
Illinois College of Agriculture, Consumer, and Environmental Sciences (ACES)
of®ce of Academic Programs and funds from the University of Illinois
Agricultural Experiment Station project number 65-0323. It was prepared as a
joint effort by several undergraduate students in conjunction with their advisor.
Any opinions, ®ndings, conclusions, or recommendations expressed in this
publication are those of the author(s) and do not necessarily re¯ect the view of the
US department of Agriculture.

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