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

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The use of genetically engineered bacteria to control

frost on strawberries and potatoes. Whatever

happened to all of that research?

R.M. Skirvin

a,*

, E. Kohler

b

, H. Steiner

c

, D. Ayers

c

,

A. Laughnan

c

, M.A. Norton

d

, M. Warmund

e

a

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

b

c

d

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.}

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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 fromÿ₃₈_{C to}ÿ₅₈_{C in natural}

samples, while distilled water samples could be supercooled from ÿ₁₂₈_{C to}

ÿ₂₀₈_{C, 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 withHelminthosporium

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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 homo-geneous ice nucleation, nuclei form spontaneously in liquid (usually below

ÿ₃₈₈_{C). 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 aroundÿ40₈C (Brown, 1997) without freezing. Water in plant tissues can be

supercooled to ÿ₅₈_{C, 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.

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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 asÿ7₈C (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.

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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).

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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

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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 nonvirulentP. ¯uorescens is reported to

overgrow and invade normal infection sites forErwinia, 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 aboutÿ₁₀₈_{C before it will crystallize.}

The mechanics of snowmaking have been reviewed (Hoffman, 1998). With

Snomax ice can form at aboutÿ3₈C (Snomax Technologies, 1999). The result is

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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 tempera-tures 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 microorgan-isms 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

Pseudo-monas strains are potentially pathogenic to humans, nonpathogenic strains of

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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. ¯uorescensA506, 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.

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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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