Ohio - Short Course 1994 Proceedings Horticulture Department Series 641 The Ohio State University Ohio Agricultural Research and Development Center Wooster, Ohio

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T · H · E OHIO SD\1E UNIVERSITY ~------~

Horticulture Department Series #641 April 1995

Proceedings of the

22"d OHIO GRAPE-WINE SHORT COURSE

February 20 - February 22, 1994 - Cleveland, Ohio

Edited by Roland Riesen

Sponsored by Department of Horticulture- The Ohio State University

In cooperation with Ohio Agricultural Research and Development Center Ohio Cooperative Extension Service Ohio Grape Industries Committee • Ohio Wine Producers Association

With the contribution of Bonnie Franks Margaret Latta Lloyd Lemmermann Judy Stetson This page intentionally blank. PREFACE

More than 150 persons attended the 1994 Ohio Grape-Wine Short Course, which was held at the Holiday Inn, Middleburg Heights, OH on February 20-February 22. Those attending were from 15 states, not including Ohio, and represented many areas of the grape and wine industry. This course was sponsored by the Department of Horticulture, The Ohio State University, Ohio Agricultural Research and Development Center, Ohio Cooperative Extension Service, Ohio Wine Producers Association and Ohio Grape Industries Committee .

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All publications of the Ohio Agricultural Research and Development Center are available to all potential clientele on a nondiscriminatory basis without regard to race, color, creed, religion, sexual orientation, national origin, sex, age, handicap, or Vietnam-era veteran status.

Water Management and Effect on Fruit Quality Diane Miller ...... 64

Yeasts in Roland Riesen ...... 68

Bacterial Considerations in Winemaking Christian E. Syberg ...... 99

Impact of Microbes on Grape Product Quality & Microbial Evaluation ofWinery Sanitation Practices Ellen Harkness ······························································-·································· 107 Influence of Wine Composition on Filtration--Diatomaceous Earth and Pad Filtration Kent R. Glaus ...... 123

Influence of Wine Composition on Filtration-Achieving Microbiological Stability Through Membrane Filtration Peter Meier ...... 128

Use of the Wine Aroma Wheel John Buechsenstein ...... 142

Lake Erie Quality Wine Alliance--Its Mission and Present Status Bob Mazza ...... 147

Can eros Quality Alliance-The Development of an Appellation Eugenia Keegan ...... 150

History of the Ohio Wine Producers Association Members and Their Role in the Ohio Wine Industry 152

Summary of the Results of the S.W.O.T. Analysis •••••.•••••..••••••••...•••••••..•••••• 153

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j j j j j j j This page intentionally blank. j j j j j j j j j j j j • j j j j j j j IDSTORY AND CULTURE IN FRANCE'S RHONE VALLEY

Karla L. Roehrig Department of Food Science and Technology The Ohio State University, Columbus, Ohio 43210

Introduction

The Rhone River appellation runs from Vienna (just south ofLyon) for nearly 140 miles along both sides of the Rhone down to Avignon. Some of the oldest in France, pre­ dating even Roman times, are located here. The region is home to distinctive and food flavored by the connections to much of Europe's most colorful and exciting historical events. Compared to the rest of France, the Rhone region is more Romanesque with a distinctive cuisine. Wines from this region remain an exceptional value both for their high quality and for cost. Somewhat overshadowed by the more popular regions of Bordeaux and Burgundy, the Rhone does not have as large a following for its earthy, spicy, bold wines. The purpose of this discussion is to examine the impact of major historical events on the culture of the area, the food and wines of the region, and the unique characteristics of and changes in its wine industry.

History and Geography of the Rhone River Valley

The source of the Rhone is from Lake Geneva in Switzerland. It joins the Saone at Lyon then flows about 140 miles to Avignon, the southern boundary of the wine appellation, and from there to the Mediterranean Sea. The region is in the south central part ofFrance (Fig. 1), but certain parts of it have only recently become incorporated into France. The river valley was a military and trade route even before the Romans came to dominate the region. Evidence suggests that many of the vineyards, widely credited to Roman expansion, actually pre-dated the Romans.

The Rhone region has been occupied for thousands of years (Table 1). There are paleolithic cave paintings dating to 20,000BC. Between 15,000 and 12,000BC, the Reindeer age, the Solutrean civilization left evidence of an advanced society which made excellent stone points. The Rhone was also occupied during the Neolithic period around 8000BC. By 2000BC, however, the Bronze age was in full flower along the Rhone river which was used to transport amber, tin and other trade goods. Advanced metalworking superior to other regions flourished here.

While the Greeks were founding Marseilles (Massalia) on the south coast of France, the Celtic princes ruled a clan-based society in the rest of France and central Europe. Although often characterized as barbaric by the Romans, this society had a prosperous and flourishing trade in wine, oils and metalwork throughout the Alps and along the Rhone and Saone rivers. Two Celtic princes founded what is today Lyon. They stopped at the confluence of the Sa6ne and Rhone rivers finding it an especially pleasant area. Seeing a flock of crows there, they named it • Lugundum (hill of crows), now called Lyon, which is today the second largest city in France . The sophistication of the Celtic society of the time is reflected in their artifacts. A particularly

1 fine example is a 5 foot tall bronze vase weighing 450 pounds which belonged to the Princess of Vix. This vase, which was likely acquired through trade with Italy, held 250 gallons ofwine and is equipped with a strainer to hold herbs that were added to the wine. That women could own such fine property and have elaborate burial sites suggests that Celtic women had considerable status and influence in their society.

In the Celtic culture dead heros were worshipped, and religious ceremonies required animal and human sacrifices. The Celts also collected the heads of those whom they defeated. Math, astronomy and the written word were all in wide use by the time the Celts were driven out of France by the Romans. The final defeat came when Vercingetorix, Prince of the Celts, lost a crucial battle to Gaius Julius Caesar in 52 BC.

The Rhone region continued to be important to the Romans. Lyon was the birthplace of Emperor Claudius. Hannibal also started his March across the Alps from Vienna. During Roman occupation of the Rhone, a number of monuments, aqueducts, spas and amphitheaters were erected, and many are still in fine shape or have been restored. In Vienna, there is a temple to Caesar Augustus and his wife Livy. Claudius, Emperor from 41-54AD, built amphitheaters along the Rhone in Lyon, Vienna, Orange and Ailes. During this time Lyon had as many as 100,000 inhabitants. Eventually the Roman civilization failed and southern France was overrun by the Visigoths from the Russian steppes.

Even though Roman civilization fell, the so-called dark ages prevailed and life was not as it was, this period still had advances, and the wine business flourished. The Christian church had extensive ties to the region. Pontius Pilate, pro-consul presiding over the crucifixion of Christ was removed from his job in the Middle East for ineptitude in office. Legend has it that given a choice between execution and suicide, he chose suicide by jumping off the cliff at Ponsas along the Rhone. Another legend suggests that he and his wife later became Christians and were martyred. Whether that was the case or not for Pontius Pilate, it clearly was the case for many early Christians along the Rhone who were slaughtered by the thousands in the amphitheaters built by Claudius. For example, Septimus Severns had 18,000 Christians slaughtered in the Vienna amphitheater alone. Not long afterward, however, the area became predominantly Christian, and the region was a focus of many crucial decisions by the early Christian church. In 529 AD, the Council of Orange met in Orange and declared predestination an integral part of Christian theology. The amphitheater at Orange is today the site of the annual Rhone wine fair in June.

The Rhone valley was also the site of the papacy during the only period when it was outside of Rome. A series of devious and no doubt illegal maneuverings led to this. Philip-the-Fair, King ofFrance, succeeded in getting his candidate for Pope elected. This was particularly important to the French King because he wanted the money that the wealthy Order of the Knights Templar had, and could only seize it with the connivance of the Pope. Thus, he persuaded Pope Clement V to bring charges against the Order even though it was highly respected. Clement V, fearing that he would be declared a heretic by Philip, carried out Philip's wishes. The Templars were officially • disbanded in 1312 by the Council of Vienna and most of the Templars were burned at the stake for heresy. Anxious that his actions would bring repercussions from Roman, Clement took up

2 residence in the walled city of Avignon in 1309. Pope Benedict XII started the actual construction ofthe Palais du Papes in 1334 and Clement VI completed it in 1352. The lifestyles of the eight Popes who lived there was often luxurious and licentious. In 13 77 Pope Gregory XI was urged to return to Rome. The French cardinals, however, were unwilling to lose control of their power base in Avignon and so elected their own Pope which precipitated the great Schism of the West which continued through the reigns of several more Popes and Antipopes. Popes battled popes. Popes killed opposing cardinals, and they all raised taxes on the peasants to support their continuing conflict. Finally, in 1409 both the Pope and Antipope were deposed, and a new Pope was elected. Until the two excommunicated Popes were forcibly removed, however, there were actually three Popes all vying for power and influence.

The Palais des Papes is an exciting attraction in Avignon. During the intervening years, it has served as a barracks and a prison among other things. It is interesting to compare the more sober older halfbuilt by Benedict XII, an ascetic Cistercian monk, with the newer half built by Clement VI who was partial, to pomp and luxury.

During the time while the Popes were in Avignon, several events altered life along the Rhone. Britain and France fought the 100 years war intermittently over several issues: Britain's claim of French land, France's desire to have a centralized monarchy located in Paris, and the control of the woolen industry. This war was particularly disruptive to the economy of the region because the wine trade was severely curtailed by the constant attacks on shipping. Their problems were exacerbated by the Black Death Plague which struck most heavily from 1347-1350. So many people were dying that Pope Clement VI consecrated the Rhone river so that the dead bodies could be dumped in it instead ofburying them. Up to three quarters of the population died depending upon the location. Throughout all ofEurope, 25,000,000 died of the plague. That level of population loss in itself caused disruption to farming, trade and the economy in general.

The next major population dislocation occurred during the . The bulk of the wealth in the region was concentrated in a few powerful families. They, of course, were executed during the fervor disrupting the fabric of society. The slaughter extended beyond the wealthy. The ofLyon in 1793 resulted in the loss of 1/3 of the population.

Hard on the heels of the revolution came a series of crises: bad crops, the potato blight and a massive flood ofthe Saone, Rhone and Loire Rivers in 1847. Three fourths ofthe children ofthat time did not reach adulthood. A telling blow to the wine industry fell softly at first in 1865 with the onset ofthe epidemic. Little did anyone at the time realize that the industry would be virtually wiped out by the end of the epidemic in the 1890s.

The Rhone river valley because of its strategic military location became extremely important in World War I. Unfortunately, vines newly replanted after the phylloxera epidemic did not fair well during the war. The loss of20% of young Frenchmen during the war coupled with increasing industrialization made labor for the vineyards scarce. With the advent of World War II, the Rhone again provided an important avenue for men and materiel. General Jean de Lettre de Tassigny's First Army secured the Rhone valley and then joined forces with General Patton's Army to give

3 the Allies their final advantage over the German forces. The center of the French resistance fighting throughout WWII was along the Rhone.

Several areas along the Rhone have become an official part of France only in the last several hundred years. Avignon was ruled as a papal legate until the French Revolution when it was returned to France. Orange and its surroundings also constituted an independent principality until the Treaty ofUtrecht united it with France in 1713.

Today the Rhone is a peaceful, picturesque area where many of the historical edifices are still extant. From the Romanesque architecture of Vienna to the medieval walled city of Avignon, a tour of the Rhone reconstructs a time travel ofEurope's most important historical events.

Wine and Food of the Rhone Valley

The wine from the Rhone appellation can be divided into two fairly distinct geographical areas: the northern Rhone and the southern Rhone (Table 2). The climates and along the river differ dramatically, and the southern Rhone has a decidedly Mediterranean climate. Subdivisions of the northern Rhone region include such famous areas as Rotie, Condrieu, St. Joseph, Hermitage, Crozes-Hermitage and Comas. The southern Rhone encompasses Chateauneuf-du-Pape, Gigondas, Tavel, Lirac and Cotes du Ventoux among others. Throughout the northern and southern regions one may find wine by the general term Cote du Rhone, but seventeen particular communes in the south are known as Cote du Rhone Villages. Although not generally capable of attaining great age, many ofthe Rhone's best , particularly in the north can easily be held for 20 years. The best ofChateauneuf-du-Pape vintages in the south will also last this long. Styles ofwine, , processing methods, and practices vary throughout the region. The grapes typically used in Rhone wines are given in Table 3. Latour has some experimental plantings including and which promise to do well. Since these varieties do not have appellation controllee status, however, wine from them must be bottled under Vin du Pays rules.

At the northern end of the Rhone region is the Cote Rotie (meaning "roasted hill"), an area near Vienna on some of France's steepest slopes. It is little changed from Roman times. Wine from here is complex, comprised mostly of and up to 20% of the white grape . The wine may need from 5-15 years of aging. Vines here are planted tepee style with three poles meeting at the top. Nearby Condrieu also relies on the temperamental viognier grape to provide a spicy . The most famous wine from this region is from the tiny vineyards ofChateau Grillet which makes only about 1000 bottles per year. Unlike the red wines of the Rhone, viognier should be consumed fairly young. St. Joseph is strung out along the west bank of the river, and both red and white wines are made here. The wines are light and fruity and not very complex. Hermitage, like Cote Rotie, is mostly made of syrrah with a small amount of white grapes added. Hermitage is on the east bank ofthe river in an area with granite soil and steep slopes near Tain. The golden, white wines ofHermitage have been characterized as rich, flinty and oily. In the same area on flatter ground is Crozes-Hermitage. The style is lighter than that of Hermitage. On down the river near Valence are the steep slopes ofthe Comas region which produces heavy,

4 tannic wines. This region has been particularly hurt by a loss of vineyard workers to industrial jobs in Valence. Also near Valence is the St. Peray area making an excellent white by the methode champenoise. There are no Rhone appellation vineyards from Valence to below Montelimar, a land scoured by the winds of the Mistral.

The most famous subdivision in the southern Rhone is Chateauneuf-du-Pape which is generally believed to refer to Pope John XXII, who was Pope from 1316-1334. Thirteen varieties of grapes are permitted in this type of wine, the blends being up to the individual vintners. Wine from this area, available in both red and white, is wonderfully rich and is often described as earthy, spicy and even gamey or barny. This was the first region in France to institute appellation controllee laws. Wine from Chateauneuf-du-Pape is very high in alcohol for several reasons. is the principle grape and is high in sugar leading to high alcohol. The soil is very rocky, trapping heat for release in the evening also promoting high grape sugar levels. Other grapes usually found in significant levels in Chateauneuf-du-Pape are mourvedre, 10% syrah and 5% cinsault. In 1991, Wine Spectator chose a Chateauneuf-du-Pape from Chateau de Beaucastel as the top wine of the year. This vineyard is owned and managed by the Perrin brothers who practice organic . Instead of sulfite, they use flash heating of the grapes to sterilize the grape skins. Some of the vintners in the area have used carbonic to enhance the fruit flavors, but this practice is falling into disfavor in this region. The best of the vintages of Chateauneuf-du-Pape are capable of aging 20 years, and many have the papal coat of arms on the shoulder of the bottle.

Another notable wine of the southern Rhone is Tavel, a rose which may contain up to 10 grape varieties. Several characteristics make this wine particularly appealing. First, unlike most roses, it is dry. The second unusual feature is its attractive slightly orange or salmon colored tint. Although this would be a fault in other roses, it is typical of Tavel and lends it visual interest. This wine is an excellent accompaniment to the lamb dishes of the region. Most ofthe vineyards of Tavel are relatively new having been replanted only in the last thirty years after lying fallow since the phylloxera epidemic.

Lirac is also a popular appellation on the west side of the Rhone. The red wines here are very light in color and best served slightly chilled. Like Tavel, many of the vines here are relatively new. Both phylloxera and WWI caused extensive damage to the vines. In fact, Chateau de Clary (still making wine) is said to be the site of the first phylloxera outbreak as the result of experiments with labrusca vines imported from the USA.

Cotes du Ventoux is south and east in the Rhone valley and uses the same blends as Cotes du Rhone. Mostly very pale reds and roses are made as the result of very short fermentation times. They should be consumed while they are young and provide a light, fruity style of wine. Clairette de Bellegard white wine comes from southeast ofNimes. The production volume is small, the wine is dry, flowery and low in acid. de Lunel is one ofthe few areas ofthe Rhone where muscat grapes are grown. They are used to produce a sweet which perhaps is not as good as Muscat Beaume-de-Venise named after a Roman spa in the region.

5 By far the largest production volume at more than one million hectoliters is wine from vineyards scattered throughout the Rhone valley and labelled as Cotes du Rhone. La Vielle F erme of this designation is widely used as a in many US restaurants. It is 40% grenache, 20% mourvedre and 10% each syrah and cinsault among others and is an excellent value, retailing at less than $1 0. Seventeen communes in the southern Rhone have won the right to label as the more exclusive Cotes du Rhone Villages.

The food of the Rhone divides into two regions as well: a butter and cream based cuisine in the north and an olive oil and garlic based cuisine in the south more typical of the Mediterranean. Some of the most outstanding food in France has its origin along the Rhone river. In fact, La Pyramide, considered to be the best restaurant in France, is located in Vienna. There are a number of signature dishes for the Rhone. The north is noted for a variety of sausages, tripe, chicken and truffie dishes. Boatmen along the Rhone are praised for a stew that is widely popular. Regional vegetable dishes that are both spicy and colorful are an excellent backdrop to the fresh fruit and honey ofthe mid Rhone area. In the south, there is excellent lamb. Every summer in Avignon, one finds the city's famous eggplant and tomato casserole. To finish a meal or as a snack, there is the extraordinary nougat ofMontelimar. The foods of the region are especially enhanced by the rich, spicy wines which accent the savory nature of the food. As one drinks the region's earthy wines and samples the outstanding food, it is impossible not to reflect on the momentous events that have shaped the history and culture of the Rhone Valley.

For Further Reading

1. Braude), F. (1991) The Identity ofFrance. Harper Collins, N.Y. 2. Caesar, G. Julius (translated by S. Handford) (1951) The Conguest of Gaul. Penguin Books, London. 3 Eleure, C. (1993) The Celts: Conquerors of Ancient Europe, H. Abrams, Inc., N.Y. 4. Fielden (1990) Travellers Wine Guide- France, Sterling Publ. Co., N.Y. 5. Rand, M. (1987) The Red Wines ofFrance, HP Books, Los Angeles, pp 88-105. 6. Suckling, J. and Gordon, G. (1991) Chateauneuf-du-Pape is rock solid in 1989. Wine Spectator, 26, #12, 22-25. 7. Tucker, A. (1989) Penguin Guide to France. Penguin Books, N.Y. 8. Ullman, W. (1972) A History of the Papacy in the Middle Ages. Methuen & Co.,London.

6 Table 1. Chronology of the Occupation ofthe Rhone River Valley.

20,000 BC Paleolithic tribes leave cave paintings 15,000-12,000 BC Solutrean civilization manufactures excellent stone points 8000BC Neolithic civilization 2000BC Bronze Age with extensive trade along the Rhone in amber, tin and metals 600BC Celts found a clan-based society with extensive trade and winemaking. Founded Lyon 52BC G. Julius Caesar defeats Vercingetorix 41 AD Claudius, Roman Emperor born in Lyon, conquered Britain 177 AD Marcus Aurelius slaughtered Christians in Lyon amphitheater and 20 years later 18,000 more were killed 800AD Charlemagne crowned Holy Roman Emperor by the Pope 843 AD France established 1309AD Philipi-the-Fair secured the election of his candidate as Pope and the papacy was moved to Avignon 1337-1453 AD 100 Years War 1347-1350 AD Black Death raged 1415 AD Papacy returned to Rome but Avignon continued to be ruled by a papal legate late 1700's French Revolution disrupted vineyards and trade 1846-1847 AD Famine, potato blight and floods cause extensive destruction and loss of life along the Rhone 1800's Lyon was the silk capital ofEurope 1865-1890 AD Vineyards destroyed by phylloxera 1914-1918 AD World War I 1939-1945 AD World War II

7 Table 2. Appellation Controllee Regions of the Rhone Valley

Northern Rhone Southern Rhone Cote Rotie Chateauneuf-du-Pape Condrieu Gigondas St. Joseph Tavel Hermitage Lirac Crozes-Hermitage Rasteau Comas Muscat -de-Baume-de-V enise St. Peray Coteaux du Tricastin Cotes du Ventoux Muscat de Lunel Clairette de Bellegard

Table 3. Grapes Often Used in Rhone Wines

Syrah Mourvedre Grenache Viognier Aubin Cinsault noir Vaccarese

8 Figure 1. Map of the Rhone River Region

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Lirac Cnateaureu1 au Paces Tavei Avignon es 0 FRANCE I~-'--'~=:::___,I

ATLANTIC OCEAN

Bay of Biscay

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9 WINES FROM RHONE GRAPES IN THE NEW WORLD

John Buechsenstein McDowell Valley Vineyard, Hopeland, CA

I am a California winemaker from Mendocino County. I have been working with "Rhone" grape varieties since about 1980 and have traveled to the Rhone Valley in France to taste wines and study wine production methods used for these grapes. I am a consulting enologist and an occasional lecturer for University Extension at U.C. Davis. Several years ago I helped found the informal group that came to be known as the "".

Today I'd like to make some comments on Rhone grape varieties grown in California, mention some viticultural peculiarities of some of these cultivars, and outline a couple of wine­ production strategies. My talk will consist primarily of slides with commentary. During the lunch that follows we will have an opportunity to taste wines made from these grapes both in the USA and in France. (NOTE: Since this lecture relied heavily on slides rather than text, this summary will list all slides shown, indicated by 0, followed by some descriptive commentary.)

0 List of major white Rhone grapes: Viognier, , , Clairette.

0 T -budded Viognier vines. The increase in Viognier grape acreage in California over the last ten years have been logarithmic: 10 years ago <2 acres 8 years ago > 10 acres 6 years ago >30 acres 4 years ago approx. 100 acres now approx. 3 00-400 acres planted

Viognier production is said to be very difficult with many associated viticultural problems: spindly canes, subject to wind damage, light-bearing with very small, tight clusters, uneven berry size at maturity (). The first Viognier crop we vinified in 1990 was only a few hundred pounds. The following slides show the "micro-vinification" production regime I followed:

0 Viognier cluster 0 Close-up ofViognier cluster showing range ofberry sizes. 0 35 lb. lug box full ofViognier clusters. Ripeness to a level of23 to 24'13rix is very important for full development of classic varietal perfume and sufficient potential alcohol to support classic flavors. 0 Transporting lugs of Viognier to the press. 0 Hand dumping lugs of Viognier to press as whole clusters. (There were too few grapes to crush. In subsequent years we did crush/destem.) 0 A stream ofjuice coming from the discharge port of the press. The juice seems to contain

10 all of the classic varietal aroma and flavor with no skin contact prior to necessary. It is important to pick and press the grapes while they are cool. 0 Transferring juice to glass carboys for cold settling. This was truly "micro-vinification" due to the small amount ofjuice. 0 Carboys half-immersed in tubs of cold water during settling. 0 Viognier fermenting in carboys. Viognier should be fermented in "passive" containers such as stainless steel, glass, or old barrels. Temperature control is an important requirement which the latter might not afford. 0 Cool fermentation temperatures maintained in this case by surrounding the glass carboys with cold water. 0 300 gallon stainless steel fermentor (specially made for the 1991 ). 0 Another view of small stainless steel tank with cooling jacket. 0 800 gallon "passive" wood foudre used at Chateau du Rozay in Condrieu for Viognier fermentation and aging. 0 Important red grapes of the Rhone: Syrah, Mourvedre, , Carignan, Grenache. 0 Close-up of a Grenache cluster. Grenache is the important "vin de base" for southern Rhone red blends. Most of the acreage in California has traditionally been planted in the hot interior valleys yielding grapes of high sugar, but low color, low acid and flavor. The tendency has been to overcrop these vines which sometimes tends to exaggerate the grape's natural tendency to "alternate bear". The best Grenache in California comes from the cooler coastal valleys grown in warm to hot days followed by cool nights. 0 Cinsaut cluster showing its large, spherical berries. This grape often contributes little color to a blend but adds perfume and finesse. 0 Syrah cluster, darkly pigmented. 0 Mourvedre cluster. This grape is very late in ripening and is often picked when the grape skin shows signs of incipient dehydration. 0 List ofvinification methods: , whole cluster fermentation, partial destemming, crushing-destemming, stainless steel only, passive aging. 0 100 year old Mourvedre vines near Calistoga. 0 T -budded vines in southeastern Mendocino County. 0 Example of the appearance ofMouvedre when ripe. 0 Close-up of ripe Mourvedre berries showing the "gold ball" puckering of the slightly dehydrated skin. 0 Mourvedre picked in small lug boxes to be introduced to a tank as whole clusters. 0 Young vines which have been t-hudded over Cinsaut. 0 Cinsaut berries at ripeness. 0 Cinsaut cluster photographed with a nickel for size comparison. 0 Cinsaut cluster pictured held in the hand. The berries are spherical and often the size of colossal olives or "shooter marbles". 0 70-year-old Syrah vines in southeastern Mendocino County trained in the gobelet fashion, commonly called "head-trained, spur-pruned". 0 8-year-old Syrah vines trained in a bilateral cordon. In France this is sometimes called "deux baguettes".

11 0 Picking Syrah into 3 5 pound lug boxes which can be manually dumped into the top of a stainless steel fermentor for "whole-cluster" fermentation. 0 Syrah grapes stacked in small lug boxes waiting to be dumped. 0 Forklifting palletized lug buxes up to tank top to be manually dumped for "whole-cluster" fermentation. 0 About 2 tons of whole Syrah clusters are dumped into a 6-ton red fermentor. No carbon dioxide is used. 0 About 2 tons ofSyrah grapes are then destemmed/crushed and pumped in on top ofthe whole clusters. A select strain of yeast native to in the Rhone valley is then added. 0 Finally, an additional2 tons ofwhole Syrah clusters are dumped in on top. 0 The whole clusters on top keep the grapes weighed down during their time in the tank. 0 Within 24 to 48 hours the active yeasts have depleted the existing oxygen in the tank and the surface is covered by a cloud of carbon dioxide.

0 The tank is sealed closed at the top to help maintain the C02 environment during the fermentation. The slow and limited availability of sugar to the active yeast generates little heat and the fermentor stays at ambient temperature (70°F). 0 The cuvaison lasts about 14 days. The little available free-run must drops to about 0°. Pump-over is not possible due to the small amount ofjuice relative to the total batch size and, in fact, does not appear to be necessary. 0 Close-up view of free-run must showing dark, brilliant color from the Syrah skins. Dark color does not indicate excessive tannins. In fact, since a majority of the berries have remained unbroken the phenolic extraction has been very gentle. 0 Unloading the tank to the press is the most difficult operation since by the end of the fermentation clusters and stems have knit together very tightly. 0 Many clusters survive nearly intact though the berries have fermented internally. 0 Pressing yields wine which still contains several percent sugar and must then be returned to the fermentor to finish primary and . 0 Best aging is accomplished in wood ofvarious sizes as long as it is "passive", i.e. used enough so as not to contribute dominant wood flavors to the wine.

The various red Rhone varieties contribute to a blend each in their own way. Grenache, the "base" wine, adds body and jam-fruit flavors, but not much color; Cinsaut adds light perfume and finesse; Mourvedre contributes a purplish-blue hue, color stability, and protection against oxidation. Syrah affords depth of color, richness, and tannic structure. All varieties seem to thrive in the warmer coastal valleys of California.

12 ADAPTATION OF THE TRAINING SYSTEM TO THE GRAPE CULTIVAR --A REVIEW OF COMMON PRACTICES

Bruce Bordelon, Extension Specialist Purdue University, West Lafayette, IN

Pruning is the most important cultural practice in the management of grapevines. It is done to establish and maintain vine shape and form, select fruiting wood and regulate the number of buds retained per vine. directly influences , fruit quality, vine vigor and hardiness. Proper pruning will result in maximum yields of high quality fruit without reduction in vine vigor or hardiness. Improper pruning will have a negative effect on each of these characters.

Training is the arrangement ofthe vine on the trellis. The purpose oftraining is: 1) to position the annual shoot growth so that the leaves receive optimum exposure to light; 2) to position the fruit for ease of pest control and ; and 3) to facilitate pruning operations. Pruning is the most expensive component of vineyard management and trellis construction is one of the most expensive components of vineyard establishment. It only makes sense to carefully consider the reasons for choosing a particular pruning and training system. The choice of the training system, pruning method and pruning severity must take into consideration vine vigor and growth habit, climatic conditions, disease susceptibility, cold hardiness, and vineyard management practices such as mechanized harvest and pruning.

HISTORY OF TRAINING SYSTEMS

Grape growing in the Eastern U.S. has its roots in New York where viticulture dates back to the early 1800's. Most of the production was of native American varieties and the training systems used were of European origin. Vines were typically pruned back to renewal zones low to the ground and shoots were trained onto stakes. American varieties were not well suited to this European style of training so new training systems were developed. Early systems followed the European principle of upright training of shoots during the growing season with arms or cordons trained horizontally close to the ground. Some of these systems such as the low wire, bilateral cordon and High Renewal are still used today for vinifera varieties.

While the upright systems were used in Finger Lakes and western New York growing regions, the training of grapes in the Hudson Valley region evolved in another direction. The well known Kniffin systems originated in this region, quite by accident. As the story goes, a vine in the vineyard of William Kniffin was crushed by a fallen limb of an apple tree. At harvest the fruit on this injured vine was noted for its large size and handsome appearance. Kniffin attributed this result to the accidental horizontal positioning of the canes. Within a couple of years a training system which we know of as the Four-Arm Kniffin was being widely used in the region. This very successful system is still widely used for American varieties today.

Several other training systems evolved from the Four-Arm Kniffin especially when vine vigor was high enough to justify leaving more nodes than could be supplied by four canes.

13 These were Six-Arm, Eight-Arm, and even Ten-Arm Kniffin systems. Another modification of the system occurred when it was observed that nodes on canes on the upper wire were more productive than canes on the lower wire. Growers began to leave more nodes on the upper canes than lower canes, and eventually to eliminate the lower canes altogether. This new system became known as the Umbrella Kniffin system, a widely used training system for American varieties today. Further modification of these training systems led to the development of the Hudson River Umbrella which utilizes cordons on the top wire of the trellis from which fruiting canes either hand down or are tied down to a lower wire. This system allows better distribution of fruiting shoots along the trellis. In recent years a modification of this system, called the Single Curtain or Bilateral Cordon system, has become popular for both French hybrids and American varieties. It differs from the Hudson River Umbrella in that 2 to 6 node spurs are retained for fruiting and renewal rather than 8 to 12 node canes. Because of the short length of the fruiting canes tying is not necessary.

The development of these training systems generally followed a pattern of increasing the height ofthe renewal zone. Sunlight exposure of the renewal region was improved as the height was increased which likely accounted for the improved productivity of these systems. It wasn't until several years after development of these systems that Shaulis, Shepard, and coworkers demonstrated the relationship of sunlight exposure to fruitfulness.

PRUNING AND TRAINING SYSTEMS

There are two basic types of pruning: cane (long) pruning and spur (short) pruning. These differ only in the length of the fruiting wood that is retained and the training method used to effectively display the fruiting wood. The concept ofbalanced pruning is used regardless of the type of pruning and training system.

There is no one system that can be applied to every cultivar and every location. Sometimes the growth habit of a cultivar lends itself better to one system than another. Some cultivars have been reported to perform better when cane pruned because the buds that are 4 to 12 nodes from the base of the cane are more fruitful than at the basal 2 or 3 nodes. This phenomenon is likely more a result of shading of the basal nodes than a genotypic trait. Proper shoot positioning should improve fruitfulness of the basal nodes and make spur pruning feasible. Some cultivars have a tendency to push many secondary and tertiary buds from canes and latent buds from cordons. Short spur (two node) pruning seems to exacerbate this problem whereas long spur (6 node) or cane pruning seems to reduce this tendency.

Vine vigor can also influence the selection of the pruning and training system. The system selected must be able to accommodate the number of buds necessary to properly balance the vegetative and reproductive growth. Vine vigor is often determined by site specific characteristics such as soils and climates as much as by the inherent vigor of the variety. High vigor vineyards frequently benefit from a horizontally divided in order to accommodate the number of buds needed to balance growth and fruiting. Systems such as the Geneva Double Curtain for downward growing varieties, and the Lyre or U system for upright growing varieties

14 are commonly used.

Grapevines are trained onto the trellis in a variety of ways. The most efficient training systems provide well spaced, evenly distributed fruiting wood along the trellis and promote full sun exposure for clusters and shoots. It is very important to expose the basal 15 or so leaves of each fruiting shoot to direct sunlight. These are the leaves that produce the sugars for the ripening fruit. Also the buds for next year's crop that form on exposed shoots are more fruitful than those on shaded shoots. Shading also has detrimental effects on fruit quality such as lower % soluble solids, higher total acidity and pH, reduced color, anthocyanin and phenolic content, and increased potassium content. Wine, juice, and dessert quality can be adversely affected by each of these changes.

Popular cane pruning systems are the Four-arm Kniffin and Umbrella Kniffin systems. These systems utilize long canes ( 10-15 buds each) that originate from renewal spurs at or near the trunk. Four to six canes are retained, wrapped around, or bent over the trellis wires and tied securely. Mechanical damage to the tender buds during the tying process can be a problem, so pruning and tying must be finished before bud swell begins. There are several major drawbacks of cane pruning systems including extra time and effort for tying, more shaded leaves, especially on the lower wires, and difficulty in leaving extra buds to protect against damage from spring frosts (double pruning).

Spur pruning systems such as the Bilateral Cordon and Geneva Double Curtain are generally better than cane pruning systems for most cultivars. In these systems two cordons from each vine extend along the top wire of the trellis in each direction. Fruiting spurs 2 to 6 nodes long are spaced along this cordon. Shorter renewal spurs are left to provide spurs for the next season. The cordons remain as semi-permanent extensions of the trunk, though they may need replacement every five or so years.

The Geneva Double Curtain system utilizes a horizontally divided canopy which is separated into two curtains of foliage. Adjacent vines are trained to opposite sides of the trellis so the cordons can extend past the adjacent vine to provide twice as much trellis space for each vine. It is particularly suited to high vigor vineyards and can result in yields up to 50% higher than single curtain systems.

In spur pruning systems the fruiting shoots should be positioned vertically upward or downward, depending on the height of the cordon and growth habit of the variety, to form a curtain of foliage. Shoot positioning is the most important aspect ofthese systems because it assures that each shoot is exposed to direct sunlight, especially in the basal regions. Sunlight exposure directly influences fruit quality and yield potential for the following year. Without proper shoot positioning, spur pruned systems fail to meet expectations.

Spur pruning systems are efficient in several respects. Selection of fruiting spurs is simplified and there is no laborious tying of canes. Good sunlight exposure results in better fruit quality and productivity. These systems are also adaptable to mechanization.

15 European (Vilis vinifera) varieties require different training systems because of their upright growth habit and lack of winter hardiness. They are trained to systems which keep the fruiting wood close to the ground to allow easy renewal oftrunks in case of winter injury, and to provide room for the vertical training of shoots to the top of the trellis. Trunks are trained to a low wire (24-36") with cordons or canes extending along this wire. Shoots are positioned vertically upward between pairs of catch wires on either side of the posts. Shoot growth is trimmed off after it reaches beyond the top wire of the trellis to prevent shading of the fruit. At least 15 leaves are retained from the last cluster to the tip to allow proper fruit ripening.

The 'spare parts' approach is often used with cold tender cultivars to assure some live buds each year. This is the Fan system in which several trunks of different ages are retained and spread out across a multiple wire trellis. Older trunks are often more susceptible to winter injury than young trunks, but are also more fruitful, so trunks of different ages are saved to provide for continuous renewal of fruiting wood. Fruiting wood is selected from canes on the different trunks. In case of winter injury to one trunk, additional buds are retained on the remaining trunks to balance the bud number.

CLUSTER THINNING AND CROP LOAD

Many French-American hybrids are highly fruitful, producing up to five clusters per shoot. Balanced pruning alone will not adequately control crop load on these varieties, especially those which produce large clusters such as Seyval, Villard blanc, DeChaunac, Chancellor and Chambourcin. On these cultivars it is necessary to remove some ofthe clusters to reduce the amount offruit to keep productivity in balance with vegetative growth. This is called cluster thinning and involves pinching off all but one or two of the developing flower clusters on each shoot. The basal clusters are usually retained because they are the largest. Cluster thinning is most effective when done just prior to bloom to promote increased fruit set on the retained cluster, resulting in more berries per cluster. Despite the removal of 2/3 or more of the potential crop, cluster thinned vines have consistently yielded as well or better than unthinned vines. In addition, fruit quality can be much better on thinned vines.

In addition to cluster thinning, many French hybrid varieties require the removal of non­ count shoots. Often both a primary and secondary shoot will develop from a single node and each will be fruitful. Additional shoots may develop from latent buds on 2 and 3 year old wood and base buds. This leads to overcropping and dense canopies. After bud break these excess shoots should be removed, allowing only the primary shoots to develop. A general rule of thumb is to retain six fruiting shoots per foot of trellis, or 20 shoots per meter. Though typically a problem, this characteristic of French hybrids can be valuable. Many varieties will develop nearly a full crop on secondary shoots after the primary buds are lost to winter injury, frost, or insect damage.

16 PRUNING TO A VOID COLD DAMAGE

Spring frost injury to grapevines is a significant problem throughout the Midwest. The situation usually arises when warm weather occurs during early spring before the average date of • the last spring frost. This promotes de-acclimation and swelling of grape buds and growth of shoots. New growth is sensitive to freezing temperatures and is damaged easily. A method to reduce spring frost damage which is adapted to spur pruning systems is "double pruning". This procedure utilizes the apical dominance of buds on a cane. The first buds on a cane to grow are those at the tip of the cane. Buds closer to the base of the cane will begin growth later than those at the tip. For double pruning, fruiting spurs are selected as in normal pruning. However, spurs are left with 10 to 15 more buds than would be called for by the balanced pruning formula. By leaving extra buds on the spurs, the buds at the basal nodes may be delayed in development by up to two weeks. After all danger of frosts is past, the extra buds are removed and the desired number ofbuds is set according to the balanced pruning formula. Delayed development ofbasal buds is dependent on weather conditions, particularly temperature. If temperatures are mild and seasonable there will be a considerable difference in the relative development of buds along the cane. If, however, the temperatures are unusually warm there will be little difference in bud development as all buds will break and begin growth at about the same time.

SUMMARY

The choice of a pruning and training system is dependent on the vine vigor and growth habit, climatic conditions, disease susceptibility, cold hardiness, and vineyard management practices such as mechanized harvest and pruning. Properly matching the training system to the particular traits of the variety and the vigor of the site can lead to improved fruit quality, productivity and ease of vineyard management. Growers should give careful consideration to the training system when establishing the vineyard. Mistakes are easier to avoid than correct.

17 IPM FOR GRAPES IN OHIO

Roger Williams, Dan Fickle, and Sean Ellis Department ofEntomology • The Ohio State University/OARDC, Wooster, OH 44691

Several species of insects and mites are pests in Ohio vineyards. Many of these pests are specific to certain regions or grape cultivars while others are endemic to all vineyards in Ohio. With more and more restrictions being placed on the usage of pesticides, the need for information that can help reduce pesticide residues at harvest and make the vineyard environment more sustainable are very important. With the support of the Ohio Grape Industries Program we have been able to study insect behavior, test new products, and develop new methods and strategies to cope with these pests. The information we have gained from this research has been assimilated into a new publication entitled, Integrated Pest Management (IPM) Insect Management Guidelines for Grapes in Ohio. There is a need to continually revise and update IPM guidelines as pests and regulations change. However, the information in this publication should be helpful in making control decisions.

The goal of IPM is to provide a commercially acceptable level of insect and mite control with minimal pesticides applied at precisely the right times. An important part of this program for grapes in Ohio is methodic vineyard inspection for these pests. In the new IPM publication, scouting techniques are emphasized. Insect behavior and biology, as well as damage thresholds are discussed. Only those insects we consider to be major grape pests in Ohio are covered in this publication, they are the grape flea beetle, grape berry moth, Japanese beetle, rose chafer, grape phylloxera, leafhoppers, European red mite, and grape root borer.

These IPM methods which have been developed for monitoring and controlling insects allow the grower more flexibility in his decision as to whether or not insecticides are needed, alternative methods of control that might be applied, and which control method to employ and when to apply it. Additional information on these and many sporadic or minor vineyard pests is available in Extension bulletins 730 and 815 (see references). Much of the potential for Rducing pesticides used on grapes will be in the area of insect control.

Once again we would like to thank the grape growers of Ohio for their support ofbasic research. This kind of support can only help keep the industry viable and at the forefront of the international grape industry.

18 REFERENCES

1. Cahoon, G., M. Ellis, R.N. Williams and L. Lockshin. 1991. Grapes: Production, Management, and Marketing. Ohio Coop. Ext. Serv. Ohio State. Univ. Bull. 815. 61 pp.

2. Ellis, M. A., and R. N. Williams. 1992. Reducing visible pesticide residue on table grapes in Ohio. Plant Pathology Dept. Ser. 90. The Ohio State Univ./OARDC, Wooster. 11 pp.

3. Ellis, M.A., C. Welty, D. Miller and R.N. Williams. 1993. Ohio commercial small fruit and grape spray guide. Ohio Coop. Ext. Serv. Ohio State. Univ. 506(B2):34 pp.

4. Williams, R.N., D. M. Pavuk and R. W. Rings 1986. Insect and mite pests of grapes in Ohio. Ohio Coop. Ext. Serv. Ohio State. Univ. Bull. 730 24 pp.

19 THE USE OF FUNGICIDES FOR CONTROLLING GRAPE DISEASES IN OHIO

Michael A Ellis Department ofPlant Pathology The Ohio State University/OARDC, Wooster, Ohio 44691

Fungicides are an important part of the grape disease management program. Due to the lack of good disease resistance in most of our currently grown varieties, combined with our environmental conditions (abundant moisture) that are highly conducive to disease development, it is my opinion that commercial grape production in Ohio is not possible without the use of "at least some" fungicide. Whereas fungicides are important, growers need to recognize that they are only ONE PART of the overall "integrated" disease management program. The effectiveness of the fungicide program is greatly influenced by use of the various cultural practices described previously and the level of disease susceptibility of the varieties being grown. For example, ifwe had a poorly pruned (dense canopy) vineyard of Chancellor grapes (highly susceptible to ) planted on a poor site (little air circulation) and with poor , the chance of any reasonable fungicide program providing an acceptable level of disease control is highly unlikely.

In addition, to use any fungicide effectively the following points must be considered:

1) Correct Disease Identification If you do not know what disease or diseases are present in the vineyard, you cannot choose the most effective fungicides for their control.

2) Selection Of The Proper Funa:icide Fungicides differ greatly in their spectrum of activity (which fungi they can control). Selection of the wrong fungicide for use on a specific disease can result in financial losses and lack of control. For example, if a grower had misidentified powdery mildew for downy mildew and had sprayed Benlate or Bayleton to control it, neither of these fungicides would have had any effect on downy mildew.

3) Proper Timina: Of Application For most diseases it takes at least a week from the time the fungus enters the plant until the symptoms develop. In the case ofPhomopsis fruit rot, the fungus enters the fruit during bloom and symptoms do not appear until the fruit begins to ripen (harvest). Depending upon the weather, it may take 2 weeks for black rot symptoms to appear. Once symptoms appear, it is too late to control the disease ~ thus, proper timing of application is critical. The fungus must be controlled before or shortly after it enters the plant.

4) Thoroua:h Coveraa:e Of All Susceptible Plant Parts Ifthe fungicide is not on or in susceptible plant parts, it can't affect the fungus. Cultural

20 practices that open the plant canopy greatly affect fungicide coverage. Proper calibration and use of the sprayer is also critical to good coverage.

FUNGICIDE USE STRATEGIES FOR CONTROLLING GRAPE DISEASE

Unfortunately, there are not many options to choose from when one considers our current fungicide use strategies. The current options are:

1) Not To Use Fun~:icides

This is always an option, but it is not the one I would recommend for commercial plantings. This option should not be confused with "organic" production. Grape growers in "organic" production systems will most probably use sulfur or copper to some extent for disease control. Sulfur and copper are fungicides. Growers that choose not to use fungicides must rely completely on cultural practices and disease resistance for disease control.

2) Protectant Fun~:icide Pro~:ram

In a protectant program, fungicides are used as a protective barrier on the plant surface. This chemical barrier prevents the fungus from entering the plant. It works much like paint on a piece ofwood to keep out water. Protectant fungicides are not systemic and cannot move into plant tissues. Once the fungus penetrates into the plant, protectant fungicides will not control it. As the protective barrier breaks down or new foliage is produced, additional applications are required to maintain the protective barrier.

Protectant fungicide programs have been, and still are very effective; however, they generally result in a fairly intensive use of fungicides. Protectant fungicides are usually applied on a 7-14 day schedule early in the growing season and on a 10-14 day schedule later in the season. Obviously, maintaining a protective barrier on the plant surface throughout the growing season requires several applications.

3) Post-infection Or Curative Fun~:icide Pro~:ram

The development and introduction ofnew "systemic" fungicides allows the use of a post-infection or curative fungicide use strategy. In a post-infection program fungicides are applied only after infection periods occur. The systemic properties of the fungicide allow it to move into plant tissues where it can stop development of the fungus after it has penetrated the plant. In the post-infection program, fungicide is applied after the initiation of an infection period, but before symptoms develop. Thus, the fungicide must be applied within 3 to 4 days (72-96 hrs) after the initiation of an infection period in order to be effective.

21 The sterol inhibiting (SI) fungicides (Bayleton and Nova) have excellent post­ infection activity against black rot and powdery mildew. Ridomil and Aliette have excellent post-infection activity against downy mildew. In dry growing seasons, with few or no infection periods, a post-infection program should result in reduced fungicide use.

Important Points To Remember About The Post-infection Pro2ram

1) In order to use a post-infection program you must be able to monitor the environment to determine when infection periods occur. IF GROWERS DO NOT HAVE THE CAPABILITY TO ACCURATELY MONITOR THE ENVIRONMENT. THEY SHOULD NOT USE A POST-INFECTION PROGRAM.

2) We need to know what an infection period is for a specific disease. This requires a great deal ofknowledge about the biology of the pathogen. At present we have this information for black rot (Table 1). We also have some predictive capabilities for powdery mildew and downy mildew, and Botrytis bunch rot. Predictive programs are currently being developed and evaluated for these diseases, and will "hopefully" be available to growers in the future.

3) Timin2 is critical. Post-infection applications must be made as soon as possible, but within 3 to 4 days (72-96 hours) after the initiation of an infection period. In order to be effective they must be applied shortly after the initiation of an infection period, and well in advance of symptom development. In most situations, once the symptoms develop, the damage is done.

Fun2icides For Controllin2 Black Rot

1) Protectants: mancozeb and ferbam are both highly effective against black rot (Table 2). Because these fungicides are strictly protectants, they must be applied before the fungus infects or enters the plant. They protect fiuit and foliage by preventing spore germination. They will not arrest lesion development after infection has occurred. I feel that mancozeb provides an excellent foundation for a protectant spray program for grapes in the northeastern U.S. It is a good protectant fungicide that will provide good to excellent control of downy mildew and Phomopsis cane and leaf spot in addition to black rot. The major problem with mancozeb is a 66 day preharvest interval (PHI) on grapes (it cannot be applied within 66 days of harvest). Mancozeb is available under many trade names and formulations. Some common trade names include: Manzate 200; Penncozeb; Dithane DF; Dithane M-45 and Dithane F45.

NOTE: It is important to note that some food processors may not accept mancozeb­ treated fruit or may have special restrictions on its use. This also applies to captan. Growers need to know where they are selling their fruit and if the buyer has any restrictions on pesticides prior to initiating a control program in the spring.

22 Ferbam will provide excellent control ofblack rot, but is not highly effective against other grape diseases. In addition, there are restrictions on the number of applications that can be used. Always read and understand the label before using or purchasing a pesticide.

Captan, Benlate and copper funa=icides (fixed copper or Bordeaux mixture) are only slightly to moderately effective against black rot and will probably not provide adequate control under heavy disease pressure.

2) Sterol Inhibitina= (SI) Funa=icides

The systemic fungicides, Bayleton and Nova, are also highly effective against black rot and will provide some post-infection (curative) activity ofthe disease if applied at the higher labeled rates and within 72 to 96 hours (3 to 4 days) after the initiation of an infection period. Post-infection or curative control must be achieved prior to symptom development on leaves or fruit. Once the symptoms are present, these fungicides will not eradicate or bum out the fungus. Both Bayleton and Nova also appear to provide good protectant activity against black rot if applied at the lower labeled rates in a protectant program. These fungicides also have excellent activity against powdery mildew.

Rubia=an is another SI fungicide that is registered for use on grapes and will provide moderate control of black rot if applied in a protectant program. Rubigan is in the same general class of fungicides as Bayleton and Nova; however, it does not provide adequate curative or post-infection control ofblack rot. Nova and Bayleton are the preferred SI fungicides for black rot control.

Funa=icides For Controllina= Powdery Mildew

Sulfur is highly effective against powdery mildew if used in a protectant program with a minimum of7 to 14 days between applications. There are many formulations of sulfur (wettable powders, dusts, dry flowables and flowables). The flowable formulations appear to be most effective and result in much less applicator exposure when preparing sprays.

NOTE: On sulfur tolerant varieties that are susceptible to PM, sulfur will be a major component of the fungicide program. On highly susceptible varieties, spray intervals shorter than 14 days (7-10 days) will probably be required.

Although sulfur is highly effective for PM control, it has little or no effect on the other grape diseases. It is important to remember that sulfur can cause severe injury on some varieties. Sulfur should only be used on varieties known to be sulfur tolerant.

NOTE: Concord grapes are extremely sensitive to sulfur. Sulfur injury may occur even on sulfur-tolerant varieties when temperatures of 80 to 85°F or higher are experienced during or immediately after application.

23 Copper fungicides (fixed coppers or Bordeaux mixture) have been rated moderately effective against powdery mildew; however, care must be taken when using copper due to the danger of foliage injury (phytotoxicity). Under heavy disease pressure, copper fungicides may not provide adequate control. I prefer not to recommend copper fungicides for powdery mildew control. However, if copper is applied for downy mildew control, it will provide some protection against PM. On less susceptible varieties such as Concord, copper fungicides will probably provide satisfactory control ofPM.

Benomyl was very effective against powdery mildew when it was first introduced; however, the development of resistant strains of the PM fungus to Benomyl have made it generally ineffective for PM control in Ohio. Where resistant strains are not present, Benomyl should provide control. I do not recommend Benomyl for PM control in Ohio.

Bayleton, Nova, and Rubigan are highly effective for control of powdery mildew. Bayleton and Nova will also provide excellent control ofblack rot, but it is important to remember that they will not control downy mildew.

FunJ:icide Resistance ManaJ:ement

The development of strains of the powdery mildew fungus with resistance to the ergosterol biosynthesis inhibiting fungicides (Bayleton, Nova, and Rubigan) is a serious threat to their continued use for powdery mildew control on grapes. There is good evidence that resistant strains have developed against Bayleton in New York, Pennsylvania and California. In order to prevent or delay the continued development of resistance, Bayleton, Nova, or Rubigan should not be used alone for season long control of powdery mildew. This means another fungicide with good activity against powdery mildew should be incorporated into the spray program at some point during the growing season. If Benomyl cannot be used due to the presence of benomyl-resistant strains, the only alternative at present is sulfur.

FunJ:icides For Control Of Phomopsis Cane And Leaf Spot

At present captan and mancozeb are the fungicides recommended for control of this disease. They are moderately to highly effective. Benlate has also been shown to be very effective.

Fungicide test results indicate that the sterol-inhibiting (SI) fungicides (Bayleton and Nova) are not highly effective. Copper and sulfur fungicides also appear to be ineffective.

NOTE: Especially where Phomopsis is a problem or a concern, mancozeb or captan should be included in the early season fungicide program.

24 Funeicides For Controlline Downy Mildew

Captan, mancozeb, Ridomil and copper fungicides (fixed coppers and Bordeaux mixture) are highly effective for control of downy mildew. All these fungicides, with the exception ofRidomil are only effective when used in a protectant spray program. They will not provide post-infection or curative activity and will not eradicate or "bum out" the fungus after symptoms appear. Ridomil is a systemic fungicide and does have curative and eradicant activity against downy mildew. Of the fungicides mentioned above, my first choice would be mancozeb. Mancozeb is the only fungicide that is highly effective against downy mildew, black rot and Phomopsis cane and leaf spot. One problem with mancozeb is that it cannot be applied within 66 days ofharvest. Even with this restriction, mancozeb remains an excellent protectant fungicide for early season disease control, and can also be used on later maturing varieties for post-bloom disease control (prior to 66 days of harvest).

Captan is excellent for downy mildew and Phomopsis cane and leaf spot, but is somewhat weak for controlling black rot. A good approach to using these fungicides for downy mildew control is to use mancozeb early then switch to captan within the 66 day pre-harvest interval for mancozeb. Currently captan does not have a pre-harvest interval for grapes.

NOTE: Although captan has no pre-harvest interval on grapes, it does have a 4-day re­ entry restriction. The following information is taken from the captan label: "Do not allow persons to enter treated areas within 4 days following application unless a long-sleeved shirt and long pants or a coverall that covers all parts of the body except the head, hands and feet and chemically resistant gloves are worn. Conspicuously post re-entry information at site of application." Remember, always read the label.

If these restrictions prevent you from using captan, then mancozeb, Ridomil and copper fungicides are your only alternatives at present.

Ridomil (metalaxyl) was registered for use on grapes in June 1993. Ridomil is by far the most efficacious fungicide available for control of downy mildew. Unfortunately, it also has a strong potential for fungicide resistance development by the downy mildew fungus. For this reason, the manufacturer (CffiA) has registered its use only as a package mix with a protectant fungicide. The two formulations available for use on grapes are Ridomil MZ58 (10% Ridomil and 48% mancozeb) and Ridomil/Copper 70W (10% Ridomil and 60% copper hydroxide). The purpose of the package mix is to delay the development of strains of the downy mildew fungus with resistance to Ridomil.

Although Ridomil is very effective, the current label use recommendations greatly restrict its use for downy mildew control in Ohio. The current labels read as follows:

Ridomil MZ58: "Make one application ofRidomil MZ58 at 1 1/2- 2lbs/A at pre-bloom only. For season-long control, post-bloom applications should be made with Ridomil/Copper, or another recommended fungicide according to label instructions. Do not apply within 66 days of

25 harvest".

Ridomil!Copper 70W- "Apply 1-2lbs. ofRidomil!Copper 70W at early bloom, l-2lbs at late bloom, and 1-2 lbs at cluster closing. Use lime with each Ridomil!Copper 70 W application according to its label and individual state recommendations. For late season downy mildew control apply other registered fungicides. Do not apply within 66 days ofharvest". Note: Other restrictions also apply. Always read the label.

Based upon the above use recommendations, Ridomil will be of limited use for downy mildew control in Ohio. In seasons when downy mildew is a problem, any post-bloom applications ofRidomil will probably be beneficial; however, additional fungicide protection will probably be required within the 66-day pre-harvest interval for Ridomil/Copper 70W. The only alternative fungicides are captan and copper fungicides.

Copper fungicides are highly effective against downy mildew and are moderately effective against powdery mildew. Copper fungicides are weak for controlling black rot. My biggest concern with the use of copper fungicides is the potential they have for phytotoxicity or "vine damage". I would prefer not to use copper fungicides on grapes if possible.

NOTE: Certain food processors, such as The National Grape Cooperative, will not accept grapes treated with mancozeb past the initiation of bloom, and the use of captan is not permitted at any time. If growers cannot use mancozeb or captan, RidomiVCopper 70W or copper fungicides are the only other chemical alternatives for downy mildew control. Thus, copper is an important fungicide for producers of processing grapes that have these fungicide use restrictions.

The best information on the use of copper fungicides on grapes that I have found is in a paper by Dr. Thomas Zabadal (Southwest Michigan Research and Extension Center) and Dr. Thomas Burr (New York State Agricultural Experiment Station). The paper is entitled "The Use of Copper and Lime on Grapes". The following summary of recommendations is intended to reduce the danger of phytotoxicity when using copper and is taken from this paper:

1. Do not make a complete season-long spray program with any copper fungicides. 2. Use fungicides other than copper whenever possible. 3. When using copper fungicides, delay their use as late in the growing season as possible. 4. When using copper fungicides, avoid the use of copper sulfate. Always use a "fixed" copper formulation. 5. Use the full recommended rate of lime. Never eliminate the use oflime completely, unless the pesticide label indicates that lime should not be used. 6. Remember that cool, wet weather enhances the risk of copper injury. Be especially certain to use adequate lime levels during such periods or switch to other fungicides. 7. Make sure that any material you tank mix with copper is compatible. Many materials are incompatible (cannot be tank-mixed) with copper. 8. Avoid copper and lime sprays on fruit destined for fresh market. NOTE: Growers

26 interested in obtaining a copy ofDr. Zabadal and Burr's paper should contact Mike Ellis.

NOTE: Aliette and ziram should be close to being registered on grapes for downy mildew control. These fungicides are highly effective for controlling downy mildew. By the time you read these recommendations, these materials could be registered. Contact your local extension specialist for information on the registration status of these fungicides.

SITUATIONS THAT MAY REQUIRE ADDITIONAL FUNGICIDE APPLICATIONS

1. Post-Harvest Applications: On varieties highly susceptible to powdery and downy mildew, some post-harvest application may be required to protect foliage and prevent premature defoliation. This is especially true on early-harvested varieties in southern Ohio.

2. Botrytis Bunch Rot: (New York State Recommendations). Use Rovral SOWP at the rate of 1.S to 2lb per acre. Botrytis bunch rot is most commonly a problem on tight-clustered French hybrids and vinifera cultivars. Proper timing and thorough spray coverage are essential for good control. Make two applications:

a) when the disease is first observed OR when the FIRST berries reach so Brix (S% sugar), which ever comes first; and

b) 14 days after the first application. A third spray may be necessary on late varieties (e.g., White ) if the interval between the second spray and harvest is greater than 4 wks. Field experience suggests that effectiveness of the fungicide is reduced following a heavy, prolonged rainfall. If such conditions occur after the last intended spray has been made, an additional application may be necessary. If only one application can be made, wait until the crop AVERAGE is S0 Brix (S% sugar). Direct the spray toward the fruit, and use a minimum of 100 gallA of water. Include a spreader-sticker, especially at the l.S lb rate.

Most growers usually try to combine or coordinate these "special sprays" for Botrytis with other "required" pesticide applications as much as possible.

NOTE: Growers in Europe and Canada have experienced loss of disease control due to the development of fungicide resistance when more than 3 sprays/year ofRovral were applied over a period of 3-S yrs. It is, therefore, strongly recommended that Rovral use be limited to a maximum of 3 applications/year to reduce the probability of developing strains of Botrytis that are resistant to this material.

NOTE: Removal of leaves around clusters on mid- or low-wire cordon-trained vines before bunch closing has been shown to reduce losses caused by Botrytis in New York and California vineyards, due to improved air circulation and improved spray penetration and coverage.

27 TABLE 1. GRAPE BLACK ROT. Leaf Wetness Duration-Temperature Combinations Necessary for Grape Foliar Infection by Black Rot

Temperature Minimum LeafWetness DurationOf for Light Infection (hrs)

50 24 55 12 60 9 65 8 70 7 75 7 80 6 85 9 90 12

Data represent a compilation from several experiments with the cultivars Concord, Catawba, Aurora and Baco Noir.

Table 2. Effectiveness of fungicides for the control of grape diseases.

Phomopsis Cane and Black Downy Powdery Botrytis Fungicide Leaf Spot Rot Mildew Mildew Rot

Bayleton 0 +++ 0 +++ 0 Benlate ++ + 0 +++ ++ Capt an +++ + +++ 0 + Ferbam + +++ + 0 0 Fixed copper and lime + + +++ ++ + Mancozeb +++ +++ +++ 0 0 Nova 0 +++ 0 +++ 0 Rovral + 0 0 0 +++ Rubigan 0 ++ 0 +++ 0 Sulfur + 0 0 +++ 0

+++=highly effective, ++=moderately effective, +=slightly effective. O=not effective. Where Benlate-resistant strains of the powdery mildew and Botrytis fungi are present, Benlate will be ineffective and should not be used. Note: The above ratings are intended to provide the reader with an idea of relative effectiveness. They are based on published data and/or field observations from various locations. Ratings could change based on varietal susceptibility and environmental conditions for disease development, or changes in fungal sensitivity to specific fungicides.

28 EPIDEMIOLOGY AND CONTROL OF GRAPEVINE POWDERY MILDEW

W. Douglas Gubler Department of Plant Pathology University of California, Davis, CA

Introduction

Grapevine powdery mildew is caused by the pathogen Uncinula nectar. Worldwide this disease accounts for millions of dollars in losses. In addition to direct yield losses, powdery mildew infection results in blemished fruit which is unacceptable or has a reduced capacity for storage. As little as 3 percent berry infection can result in off-flavors in wine and early-season infection results in berry cracking and subsequent infection by the fruit-rotting fungi , Aspergillus niger, Rhizopus spp. and Cladosporium spp.

The degree of susceptibility to powdery mildew varies. Carignane, Thompson Seedless, Ruby Seedless, Cardinal, Chardonnay, Cabemet Sauvignon and are classified as highly susceptible, while Petit Sirah, , Semillon, and White Riesling are less susceptible.

The pathogen also attacks other members of the family Vitaceae including all of the native North American grapes in the genus Vitis. These species are less severely affected than the V. vinifera varieties. Other related susceptible species are monks hood vine (Ampe/opsis aconififolia) and Virginia creeper or Boston ivy (Parthenocissus quinquefolia).

Symptoms

All succulent tissues on grapevine are susceptible to mildew infection and show characteristic symptoms. In some vineyards, especially Carignane, young shoots entirely covered with mildew can be found shortly after bud break. In the varieties Chardonnay, Chenin blanc, and Cabemet Sauvignon, initial powdery mildew symptoms have been observed to occur approximately 1 week after the first spring rain when individual colonies appear on the underside of leaves produced on the bases of shoots. These colonies give rise to secondary inoculum that result in spread of disease to other leaves and fruit in the canopy. The fungus forms a white, weblike mat ofhyphal strands (mycelium) over the infected tissue's surface. Short rootlike branches (haustoria) grow from the mycelium into the outermost layer of plant tissue to draw out nutrients. Chains of spores (conidia), borne on short stalks, arise from the mycelium, giving a dusty or powdery appearance. Mildew colonies on leaves are usually found either on the underside of exposed leaves or on both sides of well-shaded leaves. The colonies can be detected at any early stage by observation with a 1OX hand lens in the development of faint yellow patches about 6 mm (114 inch) in diameter on the upper leaf surface, and by the associated characteristic webbing and spore chains on the lower leaf surface.

29 Late in the season small, spherical, black fruiting bodies (cleistothecia) may be formed amid the mycelial mats; they contain a second type of spore (ascospore). Ascospores can act as primary inoculum for grapevine powdery mildew in California.

Disease Cycle

U. necator is an obligate parasite and cannot grow on dead or dormant grape tissue. The pathogen overwinters as mycelium in infected buds (bud perrenation) or in the form of sexually produced fruiting bodies (cleistothecia) which are washed from leaves to the bark of cordons and spurs during fall rains. Cleistothecia overwinter on cordons and spurs and release ascospores during spring rains or sprinkler irrigations.

Bud perrenation. Infection resulting from mycelium in buds occurs as shoots elongate in the spring. Mycelium grows on the surface of emerging shoots. In cool springs, shoots produced from such buds should be examined frequently for the onset of disease.

Infection often occurs on the second leaf on infected shoots and temperature plays a major role in how soon symptoms and signs are expressed. For example, at temperatures below 18°C, shoot elongation is slowed and disease onset is not evident for 14-17 days. At temperatures of 21-27°C, disease onset occurred in 7-10 days while at temperatures greater than 33°C, disease seldom occurred.

After infection, mild weather favors rapid secondary reproduction of U. necator. Spores germinate at leaf surface temperatures between 6°C and 33°C, the optimum being 25°C. Rapid germination time between spore germination and production of spores by the new colony is only 5 days. Temperatures below or above the optimum increase germination time up to 15 days. Temperatures above 3 3°C kill spores and mildew colonies.

Cleistothecia. Cleistothecia have been shown to function in the disease cycle of grape powdery mildew in New York, Hungary, Australia and California.

In California, ascospores mature in late summer and autumn and are released with summer­ autumn rains or overhead sprinkler irrigation resulting in up to two ascospore-derived germinations per year.

In the spring, ascospores are released from cleistothecia during rains and sprinkler irrigation, and are water splashed and windblown to newly emerging leaves. Individual colonies on the lower surface of the basal leaves are the first symptoms of infection and are usually visible in 7-1 0 days after the wet period that caused spore release.

Impact of Temperature. Mild weather results in increased rate of pathogen reproduction and powdery mildew growth. Conidia are disseminated within canopies and spread to adjacent vines. Germination occurs at temperatures between 6°C and 33°; the optimum temperature for growth is 25°C. At 21°C to 30°C, rapid conidial germination and subsequent production ofnew

30 conidia by the colony takes only five days.

The effect of temperature on ascospore release and germination is also highly correlative. Ascospore release occurs between 1ooc and 30°C, while no spores were released at 5°C and 35°C. Optimum temperature for ascospore release is 15°C.

Germination of ascospores is also affected by temperature with the optimum being 15°C to 20°C.

Studies show that temperature plays a greater role in secondary or conidial disease development than does moisture. Powdery mildew can develop normally over a wide range of relative humidities. However, free water in the form of rain, dew or sprinkler irrigation can reduce inoculum by causing conidia to burst or simply by washing them from the tissue to grapevine bark or the ground where they are no longer a threat.

However, conidia are somewhat hydrophobic and are not easily 'wet' by water. Many may escape the influence of wetness and water lowers the temperature under the canopy and may actually help the development of surviving infections.

Spore Distribution. The pathogen is spread by wind-born conidia. Some conidia land on grape tissue where they can germinate, produce new infection, and generate additional spores. This cycle repeats itself many times throughout the growing season.

It is unknown how far conidia can travel and remain viable or how many are produced each day. Infections increase rapidly in a downwind direction, thus, vineyards downwind from a severely infected vineyard may require extra protection.

The spread of conidia also is aided by the presence of extensive grape plantings, backyard grapevines, wild grapes, and other hosts. These areas may be the source of further mildew infection.

Control Strategies. Powdery mildew control programs work best if sulfur and/or DMI fungicides are applied as protectants. If diseases is already established before the first fungicide application in the spring, it is not advisable for dusting sulfur or the DMI compounds to be used as eradicants; instead, an application of wettable sulfur plus a wetting agent is recommended.

A general rule is that late-season control depends on early-season reduction in inoculum potential. This makes early-season control especially important to the fall-season program.

Control with Sulfur. Sulfur continues to be an effective and economical material, but in order to be effective, it must be applied before disease develops. Wettable sulfur should be applied at budbreak when dealing with ascospore infection followed by dusting sulfur at 7 to 14 days after bud break, and thereafter repeated every 7 to 10 days, until fruit begins to ripen,

31 especially in wine and raisin grape vineyards, or until the summer temperatures become too high for further infection and disease development.

During a cool, wet spring, a least one additional application of wettable sulfur 10 days after the first application is recommended; this is needed because of continued ascospore release in multiple spring rains. The wettable sulfur application is designed to eradicate initial disease, kill ascospores, and protect new foliage. Sulfuring in wine and raisin vineyards is not necessary after ripening begins, as the berries become less susceptible to mildew once they reach 8-12"Brix. To be sure the entire vineyard has reached this point, it is best to continue sulfuring until an average sugar test is 12° to 15"Brix.

Sulfur Prevents Infection by Powdery Mildew Spores. For the best control, good coverage is essential. Research has not shown whether the spores must be in direct contact with the sulfur particles for control or whether a vapor phase of sulfur is toxic. Either way, the vine must be completely covered for the most effective control.

There are some drawbacks to mildew control by sulfur application. Because sulfur washes off vines, it needs to be reapplied immediately after rain or irrigation. Immediate application is essential since optimum temperatures for mildew growth often follow rain and sprinkler irrigation.

Dusting sulfur can cause bum on vines if applied when air temperature is near 3 8°C, especially in the spring or early summer. Special care should be exercised during hot spells. Phytotoxicity can be reduced by cutting back on the amount of sulfur used or applying it in the early evening to allow slow oxidation during the night.

During season when conditions are particularly favorable for mildew development, sulfur dusting alone may not give adequate control. If a dusting is missed and infection has occurred, wettable sulfur is recommended with a suitable wetting agent and water to wash conidia from leaves and fruit. It is believed that the eradication effect is probably derived from the combination of water and the wetting agent; the wettable sulfur merely replaces sulfur washed off during the application. As in all application programs, the success of this program depends on the penetration and coverage of the vine canopy and clusters by the wash water. In severe cases, several weekly washings may be necessary for adequate coverage and control.

Sulfur/D'MI Compound Control. A more multi-faceted mildew control system is an application program combining sulfur with an approved DMI fungicide. The DMI products are locally systemic, and are not washed off by water. Unlike application with sulfur alone, there is no need for application immediately after rain or sprinkler irrigation.

One complete program includes one to three early-season applications ofwettable sulfur beginning at budbreak, followed by a DMI fungicide application at 10 to 12 inches of growth. Additional DMI applications should be made according to the instructions on the label.

32 It is important to follow label directions regarding spray intervals, concentrations, and recommended water volumes in a DMI fungicide application program. Exceeding the number of days between sprays may allow infection to occur. These fungicides decay over time and lose their effectiveness as the next spray date approaches. Stretching the days between applications beyond the recommended limit allows infection to occur before the next application.

Using more water volume in the spray tank than the label specifies may result in the vine not getting the full protective benefits ofthe product. Follow label directions regarding proper water/product ratio.

The DMI fungicides are meant to be used as protectants, not eradicants or post-infection sprays. Resistance to Bayleton (triadimefon) occurred in California populations of U necator in 1986. While problems with control occurred throughout California's grape production areas in 1986, there was little problem in 1987, a hot, dry year. However, in recent years, resistance to Bayleton in California has been a significant problem in regions where temperatures are moderate and cleistothecia function in the disease cycle. In addition, populations with high levels of resistance to Bayleton show cross resistance to Rally (myclobutanil) and Rubigan (fenarimol). Resistance to DMI fungicides also has been documented in New York, France and Portugal resulting in reduced efficacy.

While it is originally thought that reduced sensitivity to DMI fungicides would cause no crop loss, a better understanding of the effect of temperature on population dynamics and fitness of a resistant population has shed new light on how control failure occurs.

In a study conducted in California, it was found that in 19 vineyards, resistance to Bayleton occurred in 18 vineyards. One vineyard had never received DMI fungicides and is considered to be one of only a few vineyards in California to have true wild-type populations of U necator. In this vineyard the range of sensitivity to Bayleton of30 isolates was 0.1-1.9 ppm and for Rally and Rubigan was 0.1-0.5 ppm. The mean values were 0.9 ppm for Bayleton and 0.3 ppm for Rally and Rubigan. In some vineyards where resistance was documented the range of sensitivity to Bayleton was 2. 5-154 ppm. While in most vineyards surveyed, the degree of resistance was not great and sensitivity to Bayleton generally ranged from 2.5-40.0 ppm. The degree of cross resistance in each of the vineyards surveyed was much less to Rally and even smaller to Rubigan. In vineyards in which the resistance shift was severe, the population curve indicated that during the initial shift towards reduced sensitivity the population exhibited a bimodal curve during which time the range in sensitivity was extremely large. In light of the fact that resistant isolates are highly fit and tend to survive in nature very well, it becomes easier to understand how control failures occur in some years and not in others.

To fully comprehend this phenomenon, it is important to briefly review 3 factors. The first is the overwintering of resistance in ascospores. In vineyards where this has been documented, resistance in U necator colonies resulting from resistant ascospores was 22 ppm. After 5 Bayleton applications, newly produced fall cleistothecia yielded ascospores which produced colonies with resistance levels of33 ppm. Therefore, we know that resistant populations of

33 U. necator are virtually out of control before the first fungicide application is made in the spring. The second factor is temperature-influenced population increase. As stated earlier, U. necator can regenerate itself in as little as 5 days when temperatures are moderate. As temperatures decrease or increase, population regeneration time may increase to 15 days or may be stopped completely. Therefore, at a temperature of24°C the pathogen population increase may be 3 times faster than when temperatures are 30-33°C. The third factor is increased resistance with fungicide selection pressure in a given growing season. This has been documented in California; i.e., isolates may be 10-30 percent more resistant at the end ofthe season than at the beginning of the season. Disease control failures occur because disease increase in moderate temperature years or regions occurs at the maximum rate. Fungicide sprays would tend to kill wild-type isolates or those with only slightly reduced sensitivity while those isolates with greater resistance would not be entirely controlled. Isolates with high ECso values have the ability to attack grape leaves on the day ofBayleton application and these isolates would continue to increase without abatement. Under these conditions, it has been common to see control failure after the second DMI application. The reason is explainable and has been documented to be the result of highly resistant populations which become the dominant population in a particular vineyard by the middle of the growing season. Under these conditions, DMI applications using even 7-10 day intervals and full dosage will not result in economic control of powdery mildew.

Avoiding Resistance Problems. U. necator ascospores and conidia, which are resistant to DMI fungicides, are a threat in some vineyards unless a program designed to manage or avoid resistance is followed. With the build-up of documented, resistant strains in many California, New York and European vineyards, DMI compounds have lost some effectiveness. Loss of efficacy may occur if sulfur is not incorporated in spray programs using DMI fungicides.

Recent research has shown that resistance in the grape powdery mildew pathogen overwinters in ascospores, and in some vineyards, disease is initiated by DMI resistant progeny. Therefore, it is very important that information and strategies developed regarding resistance to DMI fungicides in California be followed as closely as possible if we are to save these products.

The DMI Working Group of the North American Fungicide Resistance Action Committee has developed a series of recommendations to help reduce selection pressure on the pathogen population and to preserve the full activity of the DMI compounds.

These recommendations are: 1. Do not use DMI compounds alone season-long. Use either a tank mix with sulfur or use alternating blocks of sprays with unrelated chemistry. 2. Do not merely alternate with another DMI fungicide. This will not prevent resistance development. 3. Use DMI products preventively, not curatively. 4. Thoroughly cover the crop with spray to allow penetration into the plant canopy. 5. Do not exceed the recommended maximum intervals between applications. 6. Do not exceed the permitted maximum amount applied per season. 7. Use wettable sulfur at budbreak.

34 Adhering to these resistance recommendations benefits all growers, since the development and spread into other vineyards of resistance strains affects every grower's ability to use DMI fungicides effectively.

35 INTEGRATED MANAGEMENT OF BOTRYTIS BUNCH ROT OF GRAPES IN CALIFORNIA

W. Douglas Gubler Department of Plant Pathology University of California, Davis, CA

Bunch rot of grapes () caused by Botrytis cinerea Pers. is a serious disease annually in the coastal production regions of California, while in the interior valleys this disease occurs more sporadically. The main differences between production areas are moderate temperatures and higher relative humidities in the coastal valleys. Fall rains are responsible for much of the loss in grape production. Berries are highly susceptible and fungicide applications result in minimal control because of poor coverage. While large yield losses occur during wet autumns, serious losses also may occur as the result of dense canopies or tight berry clusters. In areas where vine growth is luxuriant, heavy canopy curtaining results in high relative humidity, virtually no air movement around berries and decreased berry cuticle thickness. All of these factors result in increased susceptibility to Botrytis bunch rot. In addition, the large canopy results in poor coverage of the target site by fungicides.

Previous research has shown the benefits of grapevine canopy manipulation in Botrytis bunch rot control. As a result, growers worldwide are moving away from the old 2-wire vertical trellis and towards canopy management techniques including shoot positioning, hedging, trellis which tend to open the interior canopy to direct light and increased air movement.

CANOPY MANAGEMENT

In 1984, we set about trying to find a method to "open up" the canopy only in the area of fruit production. Research trials that year included such treatments as crown suckering, shoot removal, hedging, root positioning and leaf removal. Results showed that all treatments except hedging resulted in some degree of control, but that leaf removal provided better disease control than did three applications ofBenlate, Captan (2 lbs + 1 lb form prod/A). Hedging resulted in an increase in disease presumably as a result of secondary shoot growth and subsequent "basketing" effect in the fruit zone. Another revelation from the 1984 trial was that fungicides really had very little effect on disease incidence or severity. In 1985, trials were established in three locations to study leaf removal and fungicide applications. Trials were setup as split plot (2 x 4 factorials) with main plot effect being leaf removal or no leaf removal and sub-plot effects of single fungicide applications at bloom, preclose or with comparison to a non-treated control. Results of California trials located in Napa and lake counties are shown in Tables 1 and 2. In each trial, leaf removal from around fruit clusters resulted in significant disease control. For example, in the 1985 Napa county trial (Table 1) disease was reduced from 30.5% in the non-leafed control (NLC) to 6.2% in leafed control (LC). Fungicide applications resulted in no significant disease reduction in either leafed vines or non-leafed vines.

36 Disease was similarly reduced by leaf removal in the 1986 Lake country trial (Table 2) from 28.2% in the NLC to 5. 7% in the LC. Fungicide applications at bloom, preclose or veraison had little effect on disease, regardless ofleafremoval or not. Leaf removal on Chenin blanc vines in Monterey county in 1986 resulted in excellent disease control {Table 3). While individual fungicide applications at bloom, preclose and veraison did not further reduce disease incidence or severity, there was a significant reduction in disease when all three applications were made to NL vines.

Leaf removal studies have been conducted on several grape varieties in California, including Carignane, Chardonnay, , Cabemet sauvignon, Chenin blanc, and Thompson Seedless. In every trial, disease was significantly reduced. In addition, there were several other positive effects of leaf removal including increased bud fruitfulness, faster harvest, increased color intensity in red varieties, increased soluble solids (Brix), reduced potassium, increased T A, increased malic acid and reduced vegetativeness. The one negative associated with these studies was the reduction in yield in some trials though this too was mostly offset by reduced fungicide and application costs and cleaner fruit allowing for faster harvest.

Subsequent research has dealt with identifying reasons for the effectiveness ofleaf removal in bunch rot control. In studying the effects of leaf removal, it was determined that opening up the canopy results in several important changes in the canopy, including increased light intensity, increased temperature, decreased relative humidity and increased wind speed. In laboratory tests using wind tunnels, grape berries inoculated with Botrytis cinerea became diseased with profuse pathogen sporulation in the absence of air movement, but as little air movement as 3m/sec resulted in reduced infection and prevented sporulation ofthe pathogen. These studies confirmed that increased wind speed was the primary factor responsible for disease control though the combination of all factors resulted in conditions which were nonconductive for disease increase.

Grape berries are protected by a cuticle and epicuticular wax. Berries growing in conditions of high relative humidity, shade and decreased air movement tend to produce thinner cuticles because water loss or other stress factors are minimal. However, berries exposed to the direct sunlight immediately after berry set tend to compensate for the harsh conditions by producing thicker wax or cuticle. The cuticle is often a natural barrier to infection by pathogens and the combination of increased cuticle thickness and sub-optimum conditions for Botrytis cinerea pathogenicity result in good disease control.

Cluster architecture also plays a major role in disease. Research has shown that in grape varieties and clones which produce clusters which are full and tight, generally will have significantly more bunch rot than those with elongated rachis' with loose berries. Studies have shown the reason behind this phenomenon to again be cuticle related. It appears that when berries touch and flatten, the cuticle is formed imperfectly with areas of thin or no cuticle. This area also remains wet for longer periods of time and it is at these spots with reduced cuticle where Botrytis cinerea first attacks.

37 Because leaf removal can be costly and marginally economical, machine leaf removal was investigated. There currently are two tractor-mounted leafing machines: 1) the Gallagher Leaf Plucker and 2) the Nairns Leaf Blower. The former machine pulls leaves into a lawnmower type cage where leaves are stripped off, while the latter shreds or blows the leaves off with pulsating pressure. Both machines performed well under certain conditions. The LeafPlucker could only be used on shoot positioned vines and caused damage to shoots and canes when used on T -trellis or 2-wire vertical vineyards. The LeafBlower on the other hand, was more versatile because the head did not have to be drawn directly through the outer canopy. Both machines are being used in California and have made leaf removal more economical though many growers still pull leaves by hand.

In California, grape growers are using an integrated control program for Botrytis bunch rot. Leaf removal is practiced in virtually all premium wine-grape and table-grape vineyards. Fungicide applications are minimal in wine-grape vineyards with materials being used during bloom only if rainfall occurs with subsequent applications made only preceding rain.

Over the years, fungicides have resulted in only about 50% control on the average. Part of the reason that we do not see better control is due to poor coverage of grape clusters. This may be the result of poor equipment or vine growth, but coverage, nevertheless, is a problem. In recent trials conducted to evaluate spray coverage efficiency, it was shown that spray coverage of fruit was 200 times better in leafed vines. This sounds good, but the fact is that when compared to a cluster that was 100% covered (dipped), the clusters received 2000 times more product. Coverage problems generally can be attributed to one or more factors such as tractor speed, pump pressure, volume or concentration. Problems with any of these will result in reduced coverage which may subsequently result in more disease.

Table 1. Incidence and severity of Botrytis bunch rot of grape (. Chenin blanc) and effects ofleafremoval and fungicide application, Napa county.

PERCENT SERVERITY 1 Fungicide Application Timing2 Control Bloom Preclose Three Applications 6.2 7.1 4.0 5.1 30.5 29.2 29.2 20.1

DISEASE SEVERITY5 LR 0.38 0.43 0.14 0.27 NLR 3.36 5.14 3.65 3.06

1Percent of clusters with disease 2Benlate 1 lb + Captan 2 lb/A 3Leaf removal 4No leaf removal 5Percent root/cluster

38 Table 2. Incidence and severity of Botrytis bunch rot and effects ofleafremoval and fungicide application, Zinfandel, Lake county, CA.

DISEASE INCIDENCE' Bloom Control Bloom Preclose Veraison

LR3 5.7 5.9 3.4 6.4a NLR4 28.2 31.1 22.7 18.7b

DISEASE SEVERITY5

LR 1.2 1.0 1.1 2.9 NLR 10.7 14.2 11.2 8.2

1Percent of clusters with disease 2Rovral 2 lbs/ A 3Leaf removal 4No leafremoval 5Percent rot/cluster

39 LEAF REMOVAL EFFECTS ON BOTRYTIS BUNCH ROT AND FRUIT QUALITY IN 'VIGNOLES' GRAPE

1 2 2 Bruce Bordelon , Alan Erb and Dave Scurlock 1Purdue University, West Lafayette, IN 2The Ohio State University/OARDC, Wooster, OH 44691

INTRODUCTION

Vignoles (Ravat 51) has become a popular winegrape cultivar in recent years because of its excellent wine quality and versatility in wine style. Unfortunately this cultivar has certain viticultural limitations. It is relatively low yielding and is very susceptible to Botrytis bunch rot. Bunch rot can usually be controlled with three to four properly timed applications ofRovral (iprodione) fungicide, but this method is quite expensive and not always completely effective. There is also concern about potential development of fungicide resistance in Botrytis.

Cluster zone leaf removal is used in California and Europe to reduce incidence of Botrytis and powdery mildew, and improve fruit quality through increased sunlight exposure. The climates where leaf removal has been shown to be effective are characterized by little, if any, rainfall until the time of harvest. Climates of the Midwest are characterized by irregular periods of rainfall throughout the growing season and high relative humidity. In additional to climatic differences, most ofthis work has been done on vines trained to mid-wire or low cordon systems with shoots vertically trained and cluster thinned so that a distinct fruiting zone occurs above the cordon. Most Midwestern vineyards utilize high cordon training systems and cluster thinning is only done on cultivars that tend to overproduce. Little research has been done in this region to determine the efficacy of leaf removal in the humid climate of the Midwest. A recent study in Missouri found that the efficacy of leaf removal for control of Botrytis bunch rot varied in relation to the seasonal weather patterns and the training system used in the vineyard.

Our objectives were to determine ifbasal cluster-zone leaf removal could be successfully used to control bunch rot on vines grown under standard viticultural practices in the Midwest. Under these conditions vines are trained to a high cordon and are not cluster thinned so, consequently, do not have a distinct fruiting zone. The experiment was designed to determine the efficacy of leaf removal at various timings for control of Botrytis bunch rot. In addition, fruit quality was evaluated and experimental wines made to identify any negative effects on fruit and wine quality and yield. Successful incorporation ofleaf removal with properly timed fungicide applications could reduce disease incidence and reduce fungicide use.

MATERIALS AND METHODS

Sixty five-year-old vines of Vignoles (Ravat 51) were used in this study. The vines were trained to a six-foot-high bilateral cordon system and pruned to a 20 + 20 pruning severity using two node spurs. Fungicides were applied according to standard recommendations with the exception that no materials specifically for Botrytis bunch rot control were included. Natural

40 infection by the pathogen was sufficient for disease development. The experiment was laid out in a randomized complete block design with four replications of five treatments in three-vine plots. Leaf removal involved the removal of three leaves per fiuitful shoot from above, below, and adjacent to the basal cluster. Four different timings (treatments) were applied: Leaf removal at 1, 3, 5 or 7 weeks post bloom. An untreated control was included for comparison.

Each vine was harvested individually and the clusters separated into three subjective classes based on visual estimation of Botrytis damage (Class 1: < 10%, Class 2: 10-50%, Class 3: > 50%). Yield and number of clusters were determined for each vine/class/plot. Cluster weights were estimated from yield and cluster number data. In order to accurately determine fiuit quality, representative samples of clusters were taken for each class from each three-vine plot to determine berry weight, number of berries per cluster, disease severity(% rotten berries per cluster), 0 Brix, titratable acidity (TA), pH and volatile acidity (VA). The final plot values for disease severity, 0 Brix, T A, pH and VA were calculated as weighted averages according to the proportional weight of fiuit within each class for each plot. Plot values were then used to determine treatment means. Two years of data have been collected and analyzed.

RESULTS AND DISCUSSION

Disease severity was not significantly affected by leaf removal (LR) in either year ofthe study. Disease incidence (% clusters with Botrytis) was very high (1 00%) in both years. Disease severity(% rotten berries per cluster) was also high, ranging from 38 to 48% in 1992 and from 25 to 34% in 1993 (Table 2). Though no statistically significant differences in disease severity were detected from the weighted treatment means, there were significant differences in the proportions of the total yield that fell into the three different classes ofbunch rot severity in 1992 (Table 3). The same trend was evident in 1993, but the differences were not statistically different (data not shown). The proportion oftotal yield offiuit that was considered usable for wine (Class 1~ < I 0% rot) was 4 7% with LR at five weeks post bloom compared to 31% for the control in 1992. Though not statistically significant, LR at five weeks post bloom also had the lowest overall disease severity in 1992. Similarly, in 1993 LR at three weeks post bloom had the lowest disease severity.

Fruit quality parameters of 0 Brix, TA, and pH were not significantly different between the different arbitrary classes (Table 4). Disease severity was similar to the visual estimates of 0-10%, 11-50% and 50+%. VA also differed between classes indicating a higher degree of secondary spoilage as disease severity increased. Cluster weights decreased with increasing disease severity which was most likely due to loss of water through secondary spoilage and insect feeding. Though the fiuit quality differences between classes were obvious, when the treatment means were calculated from the weighted class values there were no significant differences between treatments for any of these parameters (Table 2).

41 Leaf removal (LR) treatments had significant effects on yield and cluster weight in 1992, and cluster weight and berry weight in 1993 (Table 1). In 1992 LR at five weeks post bloom resulted in the highest yields and cluster weights, whereas LR at one week post bloom resulted in the lowest yield and cluster weights. In 1993 LR at three weeks post bloom resulted in the highest yield and cluster weights, whereas LR at one week post bloom resulted in the lowest cluster weights and low berry weights. These results suggest a negative impact ofLR on yield components when applied at one week post bloom.

CONCLUSIONS

The efficacy of leaf removal for control of Botrytis bunch rot is difficult to determine from this data. Though there were reductions in disease severity from leaf removal, the high level of disease probably overcame the positive benefits of improved canopy microclimate through leaf removal. Leaf removal did not affect other fruit quality parameters. There were some negative effects on yield parameters with leaf removal at one week post bloom, suggesting that this timing may not be suitable. The best timing of leaf removal for control of Botrytis bunch rot without negatively affecting yield appears to be from three to five weeks post bloom.

42 Table 1. Effects of leaf removal timing on yield components. 1992

Treatment Clusters per Cluster Berry (weeks post- Yield vme weight weight bloom) kg/vine (ton/A) (no.) (gms) (gms)

0 (control) 4.20 (2.5)ab 66.3 63.8b 1.35 1 3.74 (2.2)b 62.9 59.2b 1.31 3 5.09 (3.1)ab 78.5 64.5b 1.39 5 5.52 (3.3)a 75.5 73.5a 1.32 7 4.48 (2.7)ab 71.2 62.0b 1.37

* ns * ns 1993

0 (control) 6.99 (4.2) 97.8 71.5a 1.30a 1 5.77 (3.5) 92.4 62.4b 1.06a 3 7.39 (4.4) 99.0 74.7a 1.20ab 5 6.71 (4.0) 91.0 73.9a 1.16ab 7 6.97 (4.2) 100.9 68.2a 1.12ab ns ns ** *

43 Table 2. Effects ofleafremoval timing on fruit quality. 1992.

Treatment Botrytis (weeks post) Rot TA VA bloom) (%) '13rix pH (gms/100ml) (gms/1 OOml)

0 (control) 45.7 23.4 3.11 1.61 0.033 1 42.6 23.8 3.12 1.52 0.026 3 45.1 22.8 3.14 1.46 0.034 5 37.8 22.9 3.13 1.50 0.026 7 44.1 23.0 3.10 1.51 0.026

ns ns ns ns ns

1993

0 (control) 33.7 24.7 3.26 1.21 na 1 29.9 23.5 3.19 1.20 na 3 25.2 23.5 3.25 1.21 na 5 27.1 23.8 3.22 1.21 na 7 28.2 23.3 3.22 1.18 na

ns ns ns ns na

44 Table 3. Treatment by class interactions for yield and number of clusters per vine, 1992.

Treatment (weeks post- Class 1 Class 2 Class 3 bloom) (20% rot) (50% rot) (70% rot)

Yield Clusters Yield Clusters Yield Clusters (kg/vine) per vine (kg/vine) per vine (kg/vine) per vine

Control 1.29 b 19.8 b 1.93 b 26.6 0.98 19.8 1 1.69 b 26.3 ab 1.45 c 22.1 0.59 14.5 3 1.58 b 25.5 ab 2.02 a 29.3 1.31 23.8 5 2.61 a 37.1 a 2.21 a 26.9 0.70 11.5 7 1.85 b 28.8 ab 1.63 c 22.8 1.01 19.6

* * * ns ns ns

45 Table 4. Fruit quality parameters of arbitrary classes of fiuit based on severity of rot.

1992

Class Botrytis Cluster Berry Rot(%) Weight Weight "Brix pH TA VA

1 21.4a 64.5a 1.42 22.2 3.10 1.49 0.021a 2 49.3b 73.2a 1.36 23.6 3.12 1.52 0.033a 3 70.5c 50.6b 1.26 24.4 3.17 1.56 0.040c

** * ns ns ns ns *

1993

1 14.7a 71.7a 1.17 22.7 3.21 1.21 na 2 40.8b 74.1a 1.20 24.6 3.24 1.20 na 3 66.6c 53.1b 1.13 26.5 3.27 1.20 na

* * ns ns ns ns na

46 THE GRAPE PHYLLOXERA MAKES A COMEBACK OR THE LOUSE AND THE GRAPE

Roger N. Williams Department ofEntomology The Ohio State University/OARDC, Wooster, OH 44691

The grape phylloxera is a tiny louse or aphid-like insect which attacks wild and cultivated Vilis. It is present in most grape growing areas worldwide. By the end of the 19th century, grape phylloxera had destroyed two-thirds of the vineyards in Europe. Several years ago it was discovered that the phylloxera was killing "resistant" vines in the Napa and Sonoma Valleys in northern California. It was found that vines planted on AXR# 1 were suddenly being attacked by a different "strain" or "biotype" of the phylloxera. It was known from the onset that AXR# 1 was not perfect (completely resistant) but it appeared to be the best choice and was adequate for a number of years.

This is the same louse that inadvertently had been introduced into Europe in the second half of the eighteenth century and wiped out the French wine industry. The solution was the grafting of all vinifera cultivars on American . Those rootstocks have stood for over 100 years. In California, as in Europe, the primary problem is the root form of the louse whereas in eastern North America our primary concern is the foliar form. Many insecticides have been tested to control both the leaf and root forms of phylloxera. Thiodan is currently the only acceptable insecticide for the leaf form; however, no insecticides have shown satisfactory control of the root form, to date. Yes, this is the same insect having a similar life cycle. However, in the east we must be concerned with grape cultivars which harbor the soil form allowing the insect to overwinter.

Evidence of"strains" or "biotypes" ofthe phylloxera have been demonstrated by various grape researchers (Germany: Bomer 1914 & 1924, Schilder 1947, Gotz 1962; South Africa: De Klerk 1979; Canada: Stevenson 1970; Australia: Helm 1984; Italy: Strapazzon & Girolami 1983; California: Granett et al. 1985; Ohio: Williams & Shambaugh 1988).

Ohio research of the two "biotypes" has shown that 5 of27 species of grapes (Vilis arizonica, V. jlexuosa, V. /ongii, V. monticola, and V. riparia) differ in their susceptibility to form leaf galls from two "biotypes" of the grape phylloxera, Daktu/osphaira vitifo/iae (Fitch). Electrophoretic differences were detected in grape phylloxera from six species of grapes (V. aestiva/is, V. amurensis, V. champini, V. '' (riparia x lubruscana), V. lincecumii, V. sampsonii, and V. treleasei). Clear banding differences were noted in isozymes ofleucine aminopeptidase, phosphoglucose mutase, and malic enzyme. It has been estimated that a single female could parent one billion progeny in a period of one year. This being the case, it is easy to understand the ravages of the louse on the grape. It has also been estimated that root lice in Napa and Sonoma counties may cause the wine industry to spend one billion dollars bulldozing out infested vineyards and replanting on resistant rootstocks.

47 Summary

The big question is: Will we begin to have trouble with the root form of the louse in eastern North America? Of course, this is difficult to predict but I do not believe our situation will change drastically. In the last century, people bringing vinifera --from the 'old' country into the mid Atlantic states seemed to lose the vineyard in a few years. Similar results were experienced in the Cincinnati area. In the Cincinnati area, the fall of grape production was attributed to diseases, causing the grape industry to move north. However, we now know that the grape root borer (Viatica polistiformis (Harris)) is present in the southern half of Ohio. Thus, I believe that the grape root borer was a primary factor in vineyard failures in the mid Atlantic states. Phylloxera may have played a minor role in stressing plants, but the major players were diseases and grape root borer. Whereas in California, the root form of the grape phylloxera is the most significant factor causing vineyard failure.

REFERENCES

Bomer, C. 1914. On the susceptibility and immunity ofvines to the attacks ofthe vine louse. Biol. Centralblatt. Leipzig 34:1-8. (in German)

Bomer, C. 1924. The problem of species of Phylloxera. Verh. Dtsch. Ges. Angew. Entomol. 10: 13-33. (in German)

Bomer, C. & F. A Schilder. 1934. Contributions on breeding grapevines resistant to Phylloxera and mildew. I. Introduction, II. The behavior of the leaf Phylloxera on the vines of the Naumberg Class. Mitt. Bioi. Reichsanst. 49:5-84. (in German)

De Benedictis, J. A & J. Granett. 1992. Variability of responses of grape phylloxera (Homoptera: Phylloxeridae) to bioassays that discriminate between California biotypes. J. Econ. Entomol. 85(4): 1527-1534.

De Klerk, C. A 1979. An investigation of two morphometric methods to test for the possible occurrence of morphologically different races of Daktulosphaira vitifoliae (Fitch) in South Africa. Phytophylactica. 11:51-52.

Fergusson-Kolmes, L. A & T. J. Dennehy. 1993. Differences in host utilization by populations of North American grape phylloxera (Homoptera: Phylloxeridae). J. Econ. Entomol. 86(5):1502- 1511.

Galet, P. 1979. A practical , grapevine identification. Cornell University Press, Ithaca.

Gotz, B. 1962. Research regarding the morphology of root phylloxera races in the district of Baden-Wiirttemberg. Wein-Wiss. 17:267-276. (in German)

Granett, J., P. Timper, & L. A Lider. 1985. Grape phylloxera (Daktulosphaira vitifoliae) (Homoptera: Phylloxeridae) biotypes in California. J. Econ. Entomol. 78: 1463-1467.

48 Hedrick, U. P. 1908. The grapes ofNew York. Lyon, Albany.

Helm, K. F. 1984. Phylloxera biotypes, pp. 11-13. In: G. 0. Buchanan & T. G. Amos [eds.], The biology, quarantine and control of grape phylloxera in Australia and New Zealand.· Victoria Dep. Agric., Agric. Note Ser. No. 145.

Johnson, D. T. & B. A. Lewis. 1993. Grape phylloxera (Homoptera: Phylloxeridae): comparison of leaf damage to grape cultivars grown in Arkansas. J. Entomol. Sci. 28(4):447-452.

Schilder, F. A. 1947. The length ofthe proboscis ofthe races ofthe vine aphid. Preliminary Communication. Festschr. Appel. 1947, pp. 53-54. (in German)

Stevenson, A. B. 1970. Strains of the grape phylloxera in Ontario with different effects on the foliage of certain grape cultivars. J. Econ. Entomol. 63:135-138.

Strapazzon, A. & V. Girolami. 1983. Foliar infestation ofthe grape phylloxera (Vilis vinifera (Fitch)), completing holocyclic reproduction on grafted European vines (Vilis vinifera (L.)). Redia. 66:179-194.

Williams, R. N. 1977. Augmenting early season grape phylloxera populations in an experimental vineyard. Proc. N. C. Branch Entomol. Soc. Amer. 32:38. (abstr)

Williams, R.N. & G. F. Shambaugh. 1988. Grape phylloxera (Homoptera: Phylloxeridae) biotypes confirmed by electrophoresis and host susceptibility. Ann. Entomol. Soc. Amer. 81(1):1- 5.

Williams, R. N. 1979. Foliar and subsurface insecticidal applications to control aerial form of the grape phylloxera. J. Econ. Entomol. 72:407-410.2

49 GROWING VINIFERA IN NORTHEAST OHIO

Arnie Esterer Markko Vineyards, Conneaut, OH

Introduction

Vinifera grapes grow the same as any other grapevines--no secrets. But, winter injury exists as the major problem. If growers think the winter injury problem has been conquered, think again. In 11 out of the last 30 years the winter lows have dipped below -80f, the temperature at which bud damage of about 50% occurs.

The title refers to Vilis vinifera, specifically meaning the traditional wine varieties like Chardonnay, Riesling, Cabemet Sauvignon, Pinot noir, and . The Lake Erie region should replace northeast Ohio in the title. This includes parts ofNew York, Pennsylvania and Ohio which lie along the band, five to twenty miles wide, of this great Lake's shore. Lake Erie moderates the climate as only a very large body of water can.

Finally, success depends on people--their knowledge, interest and judgment. Winegrowers must search for improved varieties and cultural practices. The following covers the priorities for survival.

Establishing a Vineyard

Survival depends first of all on a good site--the best. The best is none too good. A view of Lake Erie is almost essential. This equates to a relation between elevation and distance from the Lake. Look for moderate slope and topography which will give air and water drainage. Take soil samples. Look for structure which allows deep rooting and reasonable water perculation. Fertility should not be high.

Avoid major site modifications. Good plans will take advantage of contours. Before planting do minor grading to improve surface drainage. Lay drain tile for wet spots and problem areas. Deep plow old vineyard sites and remove old roots. However, this may not be necessary on all sites. Deep plowing also may improve the rooting zone depth and structure with topsoil and also helps early weed control. Clearing of trees around the planting may improve air movement and sun exposure. Other site modification items to consider are wind machines, irrigation, and heaters. These effect the vineyard microclimate and can be helpful with minor frost problems, but not major site defects.

For planting and trellising, lay out and plan to take advantage of contours for drainage and cultivation. We use nine foot row width for the tractors and vines 5 to 7 feet apart. Whether the rows run north-south or east-west seems insignificant. Consider how you will harvest and manage the leaf canopy.

50 Select good rootstocks and scions. For the best rootstocks, take grafts--# I 's. This means a complete callus--thumb tested, and long rootstocks--12 inches. Also, I recommend using a good diversity of rootstocks and scion clones of the cultivar you select. This is for improved wine quality and vine survival. Select propagating wood from surviving vines for winter hardiness. Avoid bad viruses and crown galled trunks.

Plant carefully and exactly on spot--lines straight and spacing even. Train trunks straight. This all helps minimize future tractor disease. Water, but do not fertilize. Keep growth moderate for hardy trunks. Let the leaves and foliar analysis indicate the need for nutrition.

Undercrop young vines. A small hand sprayer to prevent molds works wonders. Cultivate deeply to eliminate weeds and drive roots down. Mound up in the fall for winter protection and weed control. Train trunks straight using stakes, rods and strings. Straight trunks will prevent later tractor injury and cultivation problems with the grape-hoe.

Vineyard Management

Generally, good vineyard management is the same for vinifera and non-vinifera varieties. High priorities should go to practices which enhance winter hardiness. These include a balanced crop and moderate vine vigor, control ofweeds and fungal infections. Moderate stressing ofthe vines may be helpful to enhancing early wood maturity.

In pruning strive for moderate yield. Yield should be consistent and balanced year to year with vine vigor. Our goal is one gallon per vine per year (13 lbs). This is normally 40 to 50 buds on 9 x 7 spacing.

Before pruning begins and if winter temperatures have dropped below -50f, make a winter-kill study. The study should analyze primary and secondary bud kill, and the extent of injury to canes and trunks. We use five damage classes.

Class 1 - Up to seventy percent (70%) primary bud kill can be compensated by leaving extra buds and canes when pruning. Minimal cane and trunk injury should heal.

Class II - Between 70% and 90% bud kill. Leave all canes cut to about 10 buds. Look at cane and trunk damage. This is where good wood maturity will be important. A void all fertilization and minimize cultivation. Mow for weed control. This is when a large carry-over of nitrogen will overdrive the vines. The resulting bullwood makes control and hardiness difficult the next season. Sucker and trunk renewal buds need careful management; and, crown gall problems become more serious.

Class m to V - see table. This is when mounding up over the graft will pay off and provide the basis for suckers and renewal of the vines. Double trunking may be of some help for improved survival.

Other pruning practices include cutting out crown gall immediately along with winter kill. Brush cut from vines we drop in the row middle and plow under as mulch.

51 Cultivation for weed control is all mechanical or manual. The mound-up is plowed away in March or as early as pruning is done. This levels the rows for tiers, helps control weeds and leaves the floor packed in case of spring frosts. Under trellis use of the grape and hand-hoes and weed­ eater keeps weeds down. Row middles are cultivated or mowed as needed. Mound up starts mid­ August using the grape-hoe. Final two-bottom plow up follows the dormant copper spray after leaf drop in the fall.

Good canopy management can improve crop and wood maturity and make spraying more effective. To accomplish this we try to position shoots at pruning and later by combing. We try to remove suckers, do shoot and cluster thinning (Chambourcin only) by hand before bloom. Finally, hedging one to three times depending on vigor. All this to let more sun and air into the canopy and reduce fungal infection.

The spraying program avoids herbicides and insecticides because the benefits of a balance in the ecosystem seem important. Antifungal sprays start with dormant copper before bud break. Sulfur, Dithane, Bayleton and Benlate are all used as needed through bloom. Reliance on foliar feeding seems wise in vinifera to moderate vigor. Maxicrop, a seaweed extract, may help organically and contribute micronutrients. Integrating the pest management into all facets of vineyard management makes more sense all the time.

Research and the Future

Each grape and wine grower needs to do research and share it. The strength of our wine industry depends on growing at least ONE great wine grape and making at least ONE great wine. The sooner we find that grape and wine, the better. It depends on all of us.

To improve winter survival, we should make careful variety and clonal selections in combination with rootstock trials. Also, find better cultural practices and notice the little differences. Trials of microclimate modification when temperature drops below -5°F could make a vital gain in this battle. The Orchard-Rite/Kent State wind machine trial needs to be continued. Schmidlin has demonstrated the high probability of success. Especially since one-third ofthe last thirty years in the Lake Erie region had temperatures going below -8°F. The forecast for 1992- 1993 is not good.

We need to look at all aspects of management and make them work for us. This includes iasects, weeds, fungus and even viruses. Some viruses may be beneficial.

So to close, for success we need to share both our triumphs and failures to improve winter hardiness and find that special grape for this region. In addition to doing all possible in the vineyard we need to keep good records of our experiments. Sharing them helps us all progress. Our academic and research institutions play a leading and coordinating role for all ofus. But, the individual growers together contribute the majority of new ideas for basic research, and we need to keep that up.

52 Vine Evaluation Chart Winter Kill Damage Classification Vine/Damage Cat I Cat II Cat III Cat IV CatV

Primary Bud Kill to 70% 70-90% 90-100% 100% 100%

Secondary!Tertiary Bud Kill 5-10% 30-50% 80-100% 100% 100%

Cane Damage Phloem/Cambium (year-old wood) 1-2 1-2 4-5 5 5

Arm& Cordon Damage Phloem/Cambium 0 0 1-2 5 5

Trunk Damage Phloem/Cambium 0 0 0 0-1 5

Recommended PRUNE Save all Remove Cut-off Dig out pruning practice normal-- shoots-- damaged vine above vine. for each Cat. Adjust leave long arms & all graft union. Replant count with spurs (3-4). canes. Leave Leave suckers. formula. Minimum spurs (1-2). Hope vine will diameter 1/3". Suckers low. rejuvenate. Save suckers. Hope for shoots.

ADJUST FORMULA BUD CANE CORDON TRUNK Evaluation Evaluation Study Study

M=H/C * (100) (cut with S'Phloem Same as Same as cane razor) cuts in 4 cane evalua- evaluation quads base tion but on but on trunk if M =final bud All good= 1 & outwards old wood & Cordon study count Primary Grade 0-5 only if cane shows a 5 H =live bud kill= 0 for damage damage is 4-5 count wanted 2 & 3 kill = X seen (darkened C = % live buds phloem and cambium)

This summary is used at Markko Vineyard in Conneaut, Ohio in the Lake Erie viticultural region. It is based on winter proofing vines in Russia, by Vince Petrucchi and published in Wines & Vines, March and April 1981.

53 ROOT PRUNING- A POTENTIAL TECHNIQUE TO CONTROL GROWTH IN GRAPES

David C. Ferree, Alan Erb and David M. Scurlock Department ofHorticulture The Ohio State University/OARDC, Wooster, Ohio 44691

Excessive vegetative growth has been a significant problem with many perennial fruit producing plants. Excessive vegetative growth results in shading that can reduce flower initiation, fruit size, and fruit quality and thus, it must be controlled to enable sustained economic production. Over the years many horticultural practices have been used to suppress growth. Manual techniques such as bending, scoring or pruning require significant increases in labor, and chemical growth retardants have lost registration or have only been partially effective. In 1980, research began investigating the potential of root pruning as a means of mechanically controlling excessive growth of apple.

Initial greenhouse studies (2,8, 12) indicated that growth could be reduced and the greater the quantity of roots pruned, the greater the growth reduction (Table 1). Results ofthese studies indicated that the most likely cause of the growth reduction was due to stress induced through reduction in transpiration and stomatal conductance (7, 12). Subsequent work on determining the causal mechanism for the effects of root pruning indicated that reductions in sap cytokinin levels may be involved, but stress induced ethylene did not appear to play a role (13). Influence on nutrient levels in the leaves of root pruned apples was minimal (12), but fruit concentration ofP, K, and Mg were reduced and concentration ofCa increased (10).

Growth control in fruiting trees in the field was consistent (Table 1) over years (30% reduction) and resulted in improved light penetration in the canopy and a 40% reduction in pruning time (3,6,10,11). The degree of growth reduction was closely related to cropping load and growth on trees without a crop had only a 17% reduction in growth (6). With large-fruited cultivars ( 6, 10, 11, 14), yield was generally not reduced by root pruning, but in some years yields of small­ fruited cultivars can be reduced (3,6). Generally, fruit soluble solids, firmness and color were improved by root pruning and pre-harvest drop reduced. The most negative aspect of root pruning on fruiting was the general reduction in fruit size of7-10% (3, 10, 11, 14). This reduction was similar to the fruit size reduction caused by the growth retardant Alar.

Since root pruning provided consistent growth control of apple and could be applied mechanically, it appeared to be a desirable technique to try on vigorous grapevines. Certainly a reduction in growth and a corresponding reduction in pruning time, coupled with improved canopy light penetration and improved fruit quality would be very advantageous.

Reviews of the influence of root pruning ( 5, 7) reveal only a few references to the influence on grapes. Buttrose and Mullins (1) found through repeated root pruning at various levels in small­ potted grapevines that shoot length and dry weight declined in proportion to the level of root pruning. They concluded that the growth rate of shoots can be regulated by the size of the root system and suggested the effect may be due to the reduction in cytok.inins. Oniani (9) suggested that thicker grape roots regenerated better than smaller roots and regenerating capacity declined

54 with distance from the main stem. This would be just the opposite response observed in apple ( 4). A report from South Africa (15) indicates that root pruning is used in vineyards to control growth and yield. It is reported to be particularly beneficial when aisle centers are compacted and cause poor vineyard performance. The root pruning was accomplished after harvest with a vibrating plough 25 em from the vine and only one side was cut per year. They report that root pruning cause a temporary reduction in growth, followed by an extended improvement in vigor and yield.

In 1989, the tool bar on the root pruner used in the apple studies was shortened and mature vines of'Chardonnay' were root pruned on two sides, 50 em from the vine, to a depth of35-45 em at either bloom (21 June) or veraison (22 August). Due to cold temperature damage, the vines were very variable in growth and root pruning had no effect on growth or quality attributes of the juice at harvest (Table 1). It was felt that more uniform and vigorous vines would be needed to really test the potential of root pruning.

Table 1. Root pruning 'Chardonnay' in 1989 at Wooster.

Avg. Root Shoot Length Berry Pruning (em) ss pH TA wt (g)

Control 86 19.4 3.29 .91 1.60 Bloom (21 June) 77 19.9 3.27 .89 1.62 Veraison (22 August) 100 20.0 3.35 .91 1.56

In 1991, a trial was conducted in the vineyards of Chalet Debonne near Madison, Ohio. Seven replications of3 vines each ofDelaware', 'Seyval', and 'Riesling' were root pruned near bloom on two sides, 50 em from the he trunk, to a depth of 45 em. Shoot length of'Seyval' and 'Riesling' were significantly reduced by 40-45%, while growth of Delaware' was unaffected (Table 2). Although there was a tendency for a reduction in berry weight, it was not significant. There was no effect of root pruning on soluble solids, pH, or titratable acidity. Indications by the owner were that pruning time was noticeably reduced where the growth effect occurred.

55 Table 2. Root pruning at bloom of 3 cultivars at Chalet Debonne Vineyards in 1991.

Shoot Berry Length wt. ss Control (em) (g) % pH TA

Delaware 60.5 1.49 21.8 3.14 .98 Seyval 55.1 1.62 22.7 3.28 .82 Riesling 49.0 1.37 18.4 3.17 .78

Root Pruned Delaware 57.8 1.30 22.2 3.13 .95 Seyval 33.1 1.48 21.2 3.21 .71 Riesling 26.1 1.28 19.0 3.16 .79

These promising results suggested that further, more detailed research was warranted and in 1993, three studies were initiated. At Wooster on some very vigorous 'Vidal' vines, a study was established in hopes of determining the optimum time of root pruning. Vines were root pruned while dormant, at bloom, pea size, and veraison and combinations of these times. In addition to growth and yield data, measurements of photosynthesis and transpiration were made in the field at various times after pruning. Root pruning had no influence on any aspect of growth or yield.

'Catawba' and 'Seyval' vines at Kingsville were also root pruned either at bloom or veraison. Growth or juice quality at harvest were not influenced. The lack of results from these three studies was very disappointing. In order to attempt to understand why root pruning had little effect, a greenhouse study will be initiated this winter to see the quantity of roots that must be removed to influence growth. Treatments in the field will be adjusted based on these results to see if this technique can be used to reduce growth of vigorous vines.

REFERENCES

1. Buttrose, M.S. and M.G. Mullins. 1968. Proportional reduction in shoot growth of grape vines with root systems maintained at constant relative volumes by repeated pruning. Austral. J. Bioi. Sci. 21: 1095-1101.

2. Ferree, D.C. 1989. Growth and carbohydrate distribution of young apple trees in response to root pruning and tree density. HortScience 24(1):62-65.

3. Ferree, D.C. 1992. Time of root pruning influences vegetative growth, fruit size, biennial bearing, and yield of 'Jonathan' apple. J. Amer. Soc. Hort. Sci. 117(2): 198-202.

56 4. Ferree, D.C. 1994. Root pruning in root distribution of'Melrose'/M.26 apple trees after nine years of root pruning. HortScience (in press).

5. Ferree, D.C., J.R. Schupp, and S.C. Myers. 1992. Root pruning and root restriction of fruit trees--current review. Acta. Hort. 322: 153-166.

6. Ferree, D.C. and W.T. Rhodus. 1993. Apple tree performance with mechanical hedging or root pruning in intensive orchards. J. Amer. Soc. Hort. Sci. 118(6):707-713.

7. Geisler, D: and D.C. Ferree. 1984a. Response of plants to root pruning. Hortic. Rev. 6:155-188.

8. Geisler, D. and D.C. Ferree. 1984b. The influence ofroot pruning on water relations, net photosynthesis and growth of young 'Golden Delicious' apple trees. J. Amer. Soc. Hort. Sci. 109:827-831.

9. Oniani, D.V. 1973. The effect of severing grapevine roots on their regeneration. Hort Abstr. 45:1459-1974.

10. Schupp, J.R. and D.C. Ferree. 1987. Effect of root pruning at different growth stages on growth and fruiting of apple trees. HortScience 22(3):387-390.

11. Schupp, J.R. and D.C. Ferree. 1988. Effects of root pruning at four levels of severity on growth and yield of'Melrose'/M.26 apple trees. J. Amer. Soc. Hort. Sci. 113(2): 194-198.

12. Schupp, J.R. and D.C. Ferree. 1990. Influence oftime ofroot pruning on growth, mineral nutrition, net photosynthesis and transpiration of young apple trees .. Scientia Hort. 42:299- 306.

13. Schupp, J.R. and D.C. Ferree. 1994. Effect of root pruning on shoot tip ethylene production and xylem concentrations of cytokinin and 1-aminocyclopropane-1-carboxylic acid in young apple trees. OARDC Res. Circ. (in press).

14. Schupp, J.R., D.C. Ferree and I.J. Warrington. 1992. Interaction of root pruning and deblossoming on growth, development and yield of 'Golden Delicious' apple. J. Hort. Sci. 67(4):465-480.

15. Vanzyl, J.L. and L. Van Huyssteen. 1988. Root pruning in farming in South Africa. Vorl 216:1-2.

57 Table 3. Summary of the influence of root pruning on apple over 10 years of research and growth experience in Ohio.

Physiology Change compared to not root pruned (%)

Photosynthesis -11% to -23% Transpiration -58% Water Potential +43% Stomatal conductance -68% Shoot ethylene -49% ACC-Conc. NS Sap cytokinin -51% Shoot and leaf carbohydrate NS Root carbohydrate -30%

Nutrition Leaf Fruit Nitrogen NS Phosphorus NS -24% Potassium NS -22% Calcium NS +7% Magnesium NS -17%

Growth Shoot -30% Trunk area -53% Leaf -5% Roots 10 mrn dia. -55% Pruning time -40% Canopy light +10 to 50%

Fruit Effects Fruit size -7 to -10% Preharvest drop -50% Fruit color +16 to +35% Firmness +6% spot -45% Russet -30% Soluble Solids +11% Yield NS to -14% Biennality -50%

58 CONFERENCE NEWS AND EQUIPMENT UPDATE

Andrew Wineberg The at WolfCreek, Norton, OH 44203

You would not be here today ifyou did not believe in continual education and change. That's why we attend conferences, that's why we sit through presentation after presentation, and that's why we succeed as grape growers and winemakers. We, in Ohio, are in an industry very much in flux; our growing techniques and our vinification styles shift constantly. We are not a static industry -- we are always searching for ideas and means for improvement. Innovation and change are essential.

There are two ways to come up with innovative ideas for change: by experience and hard work, or, my favorite, by stealing the ideas from somebody else. I would guess this intellectual theft is why most of us attend these conferences.

I'd like to give a quick thieve's report on last summer's Pruning Mechanization and Crop Control Symposium in Fredonia, NY, and the following Eastern Section meeting of the American Society for Enology and Viticulture, held in Rochester. I'll also talk a bit about equipment.

The two-day Rochester meeting featured a lot of research papers and a lot ofwines. It is always interesting to taste what other states are doing with "our" varieties. It becomes very clear that Ohio is often leading the pack -- our wines are better than we realize. Other states are, no doubt, stealing ideas from us.

I did not extract much useful information from a talk on skin fermentation of red Muscadines, or from the talk on the incidence of grapevine yellow disease in Virginia. My ears perked up, though, with three different papers on Seyval, numerous papers on various yeasts, and others.

Particularly useful to me was a Michigan paper by Smithyman and Howell on canopy configuration with Seyval, and another paper they presented, also on Seyval, looking at carbohydrate sink competition. If you grow Seyval, read these papers.

I'm not here to provide a synopsis. The papers are published in the American Journal of Enology and Viticulture, and are available to you. I would like to encourage you to join the Eastern Section, and attend the annual meetings. You should note that the next annual meeting will be in this building, in mid-July 1995.

At the meeting, a luncheon provided by Gist Brocades spotlighted various California wines side-by-side treated with their AR-2000 enzyme, which is designed to express fruitiness in the wines. We thought some of the wines were improved, so we did some work at our winery with the AR-2000 enzyme. In our tasting panels, not a single one of our wines was improved by using this enzyme. However, I am sure the enzyme will find use improving insipid, flavorless wines from other states.

59 Obviously, not all ideas you steal prove useful. At the Fredonia symposium the main focus was Concord -- not something I normally find interesting. But keep in mind that Concord is probably the least economic grape you can grow. It suffers from low prices and aging vineyards. The growers who are surviving are surviving through innovation, inventiveness, and ingenuity.

They are a shining example for all of us.

We spent a lot oftime out in the field, both in the research vineyards and in private vineyards. A lot of work is being done to reduce the labor demand in Concord, and we were looking at vineyards that were pruned with hedgers, or summer pruned, or not pruned at all. A "minimally pruned" vineyard is a sight to behold, sort oflike no-till com but uglier.

Over several years, the minimally pruned Concord vines, if you view them from the end of the row, have the outline of a mushroom. The massive tangle of canes in the top mat go against everything you have ever learned about canopy management. But the vine makes allowances. After a few years the vine stabilizes its growth pattern, and the number of clusters is very high -­ but the number of berries per cluster drops; the number of shoots increases -- but the shoot length drops; the interior light levels drop -- but the canopy surface area increases. All this, with similar yields and sugar contents. Keep in mind, these Concord growers are professionals, do not try this at home with your !

Grape growing is hard work, and we cannot always count on selfless, cheap vineyard labor. Labor saving innovations for Concord, because of the tricky economics, are imperative. Some growers have mounted virtually every piece of equipment they have on over-the-row harvesters, and they use the machines throughout the year. Shoot positioning systems, designed to open up the canopy for better light penetration, were much in evidence. Tractor mounted cutter-bar hedgers are commercially available for pre-pruning and summer hedging, and harvester-mounted herbicide and canopy sprayers were available for inspection.

Wine grapes demand different growing techniques, and much of the experience and inventiveness of Concord growers do not apply. Those of us in wine grapes who play around with alternative trellising systems and vine management are much more interested in subtle fruit quality parameters, and not so much interested in massive quantities of $200 a ton juice grapes.

If we are interested in row width, and intensive planting, we like to look at smaller tractors. If we have specialized vines grown in special ways we need special equipment. If we have tightly aggregated rock 15 inches down which prevents setting posts, we have to rip out some root room with a D-9 bulldozer and come up with self-supporting trellis systems.

We can take advantage of cheap local materials, and perhaps manufacture on-site concrete posts, or cut our posts in the local swamp, or dispense with posts entirely. If we really have time (and money), we can do everything right, even to the point of discouraging rabbits. There are a lot of tempting ideas out there, and it is important that you not be afraid to steal them.

60 Beyond the growing of quality grapes is the production of the quality wine. We all have opinions about production equipment. One winery will show you the very best tank design, which is quite different from the tanks at the winery down the road, whose winemaker looks down on the poor tank design of the previous winery. Another winery's specialized tank is obviously a much improved design over the previous winery's design. There seems to be some subjectivity in tank design parameters.

I have my own opinions about equipment, which you may take as gospel. Crushers are very important. Do not bother to grow a quality grape if you are going to beat it to death in a poorly designed de-stemmer. Presses are also critical-- you need to be able to control oxygenation, and pressing pressure. And don't treat every grape the same.

Fermentation temperature control is absolutely essential. This does not have to be overly expensive. Many wineries are quite ingenious in cool fermentation technology.

A place you should spend money would be the bottling line. How you treat the wine when you bottle makes a difference. We have also found, even in a small operation like ours, that the labor savings in a well engineered packaging line will pay for the machinery in four or five years. Look at your actual costs. Think about it. There are products, though, that tend to be labor intensive, and not easily mechanized.

In the Ohio wine industry, where we have problems, there are solutions. We need to get better at producing, consistently, quality grapes. We must look beyond locally traditional systems of vineyard management, and not be afraid to try to piece together systems using innovative ideas. We, at some point, must define what might make an Ohio wine unique-- and then we must market that unique product beyond state lines. In every winery, we need to make an effort to properly equip ourselves so that we do not routinely accept quality trade-offs in our wine production. It does not make sense to wait until you sell more wine, to make a better wine.

Figure out what kind of a grape, or what kind of a wine, you really want to produce. Go to other growers, go to researchers, go to France; wherever you go, ask questions and steal ideas. Take the products of your theft and be ingenious, incorporate them into your system, and make it work. Ifyou want, paint their ideas in your colors and call them your own. This low risk thievery has a great pay-back, and every wine in Ohio should proudly wear a sticker saying "Quality wines made in Ohio by not-yet-indicted Ohioans."

61 CONFERENCE NEWS AND EQUIPMENT UPDATE

Nick Ferrante, General Manager Ferrante Winery, Geneva, OH

Wineries Unlimited held at Adam's Mark Hotel, Philadelphia, Pennsylvania on February 9 -12. This seminar is sponsored by Vineyard and Winery Management Magazine.

Various seminars ranging from fruit wines, barrel effects, winemaker workshops, white hybrids as an alternative to Chardonnay-style wines, marketing and tourism were held.

The fruit-wine seminar which is held yearly, featured blueberry wine. Understanding how to make and market blueberry wine was covered. East-coast producers were highlighted and shared their expertise.

A barrel effects seminar was held to discuss phenolic uptake during fermentation and wine storage. The phenolics were analyzed during pre-fermentation or maceration stage, during alcoholic fermentation and post-fermentation. Pre-fermentation maceration extracts anthocyanins, the alcoholic fermentation extracts anthocyanins and tannins and the post-fermentation allows structural changes of the anthocyanins and tannins. This talk was presented by Jacques Recht of Ingleside Winery in Virginia.

A tasting was held to distinguish stylistic differences between French and American oak, both heavy toasted. One barrel was planed (one-fourth inch taken off inside the stave) and one was unplaned. Wines tasted from these barrels were a California Pinot Noir and . Barrels were coopered by Independent Stave Co. I didn't taste much difference in the planed and unplaned wine samples. When asked why the barrels were planed, the response was for stylistic choice.

The first Winemaker Workshop featured Riesling with an East and West coast perspective. David Munksgard (Glenora Wine Cellars, East coast perspective) stated that skin contact, slow and cool fermentation ( one-half 0 Brix decrease per day), stopped fermentation around 2° Brix, blending wine from different vineyards and early bottling are his key elements in making Riesling.

Peter Bell, winemaker at Vinifera Wine Cellars in New York, felt that separating press fractions, a slightly warmer fermentation around mid 60's and juice reserve blending were his key elements.

Dennis Martin from Fetzer Winery, California looks for yearly uniformity. Yields in Monterrey County range from 4-6 tons per acre. The juice is settled, centrifuged, bentonited (4-6 #/1,000 gal.) and cool fermented at 48 to 50°F. The fermentation is stopped around 3°Brix.

62 Slightly different treatments offer stylistic choices that result in semi-dry . Final result - all sell very well with little marketing efforts.

The second Winemaker Workshop featured East and West coast perspectives on .

David Lake winemaker at in Washington state, noted that vineyards in Washington are inland, not coastal in comparison to California. This provides good water and air drainage. Another factor was that most vineyards are hillside locations. Irrigation controls growth in the vineyard at key times. He states that many wineries in Washington are pressing Merlot early and finishing the wine in oak. This gives them good fruit retention and good oak balance.

Alan LeBlanc-Kinne from Virginia states that the vineyard site selection is the key to survival of Virginia Merlot. With Virginia's humid climate, leaf pulling and use of the Scott Henry trellis helps relieve disease pressure. Le-Blanc-Kinne is using , up to 20% as a blending tool. Barrel fermentations with red vinifera are also being researched.

Dan Kleck, winemaker at Palmer Vineyards on Long Island, New York, also states that site selection and soil drainage are important to wine quality. Kleck feels that rootstock selection helps the wine ripen earlier and increase winter hardiness. Long Island producers are blending , 15 to 25%. He also states that adjusting a high pH (e.g., from 3. 7 to 3 .4) makes tannins harder, and spoilage will not occur.

The American Society for Enology and Viticulture - Eastern Section (ASEV-ES) sponsored a seminar about making white hybrid wines in a Chardonnay-style. Dr. Jim Gallander from Ohio State University gave a talk that featured white hybrid wine production. Stan Howell, from Michigan State University gave a viticulture talk about growing white hybrids and how to select the correct vineyard practices. Various wines made from Vignoles, Seyval and Cayuga were tasted. Most wines had barrel treatments similar to Chardonnay production. I think it is easy to make this style of wine, but marketing these wines away from the winery is a harder task to accomplish.

The equipment trade show at Wineries Unlimited is the biggest collection of vineyard and winery equipment and accessories in the Eastern United States. Winery equipment present: crushers, presses, pumps, tanks, filters (diatomaceous earth, plate and frame and lees), bottling lines and labelers. If you are considering buying equipment in the future it is nice to see everything under one roof and talk to the representatives from the companies.

63 WATER MANAGEMENT AND EFFECT ON FRUIT QUAITY OR WHY DO BERRIES SPLIT?

Diane Miller Department ofHorticulture The Ohio State University/OARDC, Wooster, OH 44691

During the growing season, water, temperature and sunlight are the major environmental regulators of grapevine and berry growth. These three major regulators can be modified only slightly by our cultural practices. Variations in moisture, temperature, and sunlight among seasons and among regions greatly affect berry quality, and consequently wine quality. The year to year variability leads to "vintage" or "not-so-vintage" years for wines produced from grapes grown in our unpredictable environment.

Regulating water can regulate vegetative growth, berry size and berry composition. Ohio receives plenty of rainfall to produce high quality grapes - but often the rainfall isn't received when it is needed. Ideally, the vineyard is supplied with approximately "one-acre-inch" (roughly 27,000 gal.) ofwater per week throughout the season. When there is a deficit or excess of several inches of water throughout the season, as we have seen in recent years, this is a large amount of water to add (by irrigation) or remove (by drainage).

The competitive disadvantage the Midwest has in grape growing and wine production is unequal rainfall distribution throughout the season and reduced light. From this Midwest perspective, rainfall adds water but reduces light while clear skies add light but reduce moisture levels. The "normal" fluctuation between clear skies and rainfall works fine, and is productive but variable. Extended periods of cloudiness greatly reduce light availability and production of sugars via photosynthesis by plants.

Midwest growers need to be excellent vineyard managers of moisture (either excess or inadequate) and of light (always inadequate). The better these factors can be managed or controlled the higher the chance for quality wine produced from grapes grown in these vineyards.

In areas of the world where moisture can be controlled since rainfalls are predictable and most growing season moisture is added by irrigation, irrigation can be used as a to9l to ensure quality grapes. For example, in Australia, researchers are working with grapes to: 1. identify compositional and yield objectives; 2. identify physiological processes of vines which influence compositional characteristics and yield components; 3. ascertain how these processes are influenced by vine water relations; 4. determine and define optimum seasonal pattern of vine water relations which will achieve the objectives; and 5. define the pattern of soil water status which will optimize vine water relations. In theory, at least, this will lead to uniformity from year to year­ and high quality.

64 In the Midwest, moisture for vine growth is dependent upon: frequency and amount of rain; rate loss of water from the vineyard by evaporation, transpiration and drainage; and the amount of water the soil will hold to which plants have access. To obtain maximum control over vineyard moisture in the Midwest, growers should select a medium-textured soil, tile well (to drain), and be able to irrigate (with trickle, ifjust moisture and not frost-control is being considered).

Moisture-related effects are not identical in vineyards across the Midwest because of differences in soils, grape root systems and varieties, and crop loads. Moisture excess is often compounded by reduced light availability. Moisture deficit occurs as plants transpire and soil moisture is lost by evaporation. As a critical soil moisture deficit is reached, plant stomates close thereby reducing transpiration (and photosynthesis!). Unless water is added by irrigation (or rainfall), grape yield and quality are reduced.

Plant physiological processes affecting vegetative growth in the current year, fruiting in the current year, and fruiting in the following year occur simultaneously during the growing season. Moisture deficiency or excess will affect all three processes at any time during the season. Whether this results in an economic impact depends upon timing and severity of the stress.

Moisture greatly affects grape berry growth potential and actual growth. Berry growth occurs in a double sigmoid pattern where early logarithmic growth is due to cell division, seed maturation (fruit color change) occurs while logarithmic growth ceases, and then logarithmic growth occurs again due to cell expansion by water uptake leading to berry ripening. In 1993, Ohio had good soil moisture before and during bloom. Limited and scattered rainfall occurred during mid-season, the time of berry cell division. The number of cell divisions determines maximum berry size and increased water stress resulted in fewer cell divisions and consequently smaller berry size potential.

Small berry size is not necessarily bad as smaller berries have a higher skin to flesh ratio and it is the berry skin which supplies color and flavor compounds to wine. In fact berry size and light exposure are regarded to be the two major factors affecting wine composition. However, small berry .size potential can result in skin splitting if high levels of moisture are available throughout the cell expansion stage.

Let us consider the relationship between moisture, berry development and skin splitting. An unripe (pre-veraison) grape berry is hard, green, acidic and unsweet. Such a berry also has completed cell division and further size increase is due to cell expansion. At veraison, berries begin to soften, the skin changes color, there is rapid sugar accumulation, and degeneration of xylem function occurs (which means major import into berries is via phloem sugar water). After veraison, berry volume begins to increase rapidly.

The parts of the berry are the skin (including waxy cuticle), flesh, seeds, and vascular connections. Four distinct water flows are involved in berry development: xylem and phloem intake through the stalk, transpiration from the skin, and osmotic uptake (inhibition) through the skin. When inflows of moisture exceed outflows, the berry swells. This usually occurs during the

65 night. When outflows exceed inflows, the berry shrinks. This usually occurs during the day. Net growth over time is the result of small excesses of swelling over shrinkage.

Berry splitting is a water relations problem causing stress:strain between berry flesh and skin. Environmental factors involved in berry splitting are wet or humid weather and soil water. In experiments on fruit expansion (for fairly mature, nearly full grown fruit), under dry conditions, outflows almost exactly balance inflows with little net daily size increase. However, under cool, humid, cloudy weather (i.e. conditions which lessen transpiration), there was an eight-fold daily size increase as compared to dry conditions. Under wet skin conditions (dew, condensation, rain), there was a twenty-five-fold daily increase as compared to dry.

To avoid splitting, skin stretchiness at each stage of development must be sufficient to accommodate the short periods of rapid expansion growth .brought about by wet or humid weather. Experimentally it has been shown that an initially soft berry can survive wet conditions for about four days before skin bursting. An initially more turgid berry can tolerate wet for considerably less time before splitting. A berry with a slightly imperfect skin can tolerate wet for less time before splitting. The surface: volume ratio of small berries is higher and more sensitive to changes in berry volume than that oflarge berries. As an analogy, it is easier to pop a small balloon blowing it up than it is a large balloon. The stage was set in 1993 for berry splitting: there was water stress and reduced cell division and cell number, and there was lots of moisture for long periods of time at harvest.

Berries do have some feedback mechanisms to prevent skin splitting including a reduction in phloem sap inflow with increased berry turgor; increase in transpiration outflow; and a reduction in osmotic water uptake through skin as the berry stays wet. An additional mechanism in some varieties (e.g. Muller-Thurgau) is sap extrusion through lenticels on the skin as a safety valve. Sometimes however these mechanisms are overtaxed and the skin splits.

Berry splitting occurs in many fruits including grapes, cherries, apples and nectarines. There appears to be a genetic component to the characteristic as the tendency is cultivar-dependent. In all cases, fruit splitting results in a loss of quality, value, and utility. Additionally, the way is opened for secondary damage such as drying, and invasion by pathogens.

66 References:

1. Hrazdina, G., G.F. Parsons, and L.R. Mattick. 1984. Physiological and biochemical events during development and maturation of grape berries. Am. J. Enol. Vitic. 35(4):220-227.

2. Lang, A. and H. During. 1990. Grape berry splitting and some mechanical properties of the skin. Vitis 29:61-70.

3. Lang, A. and H. During. 1991. Partitioning control by water potential gradient: evidence for compartmentation breakdown in grape berries. J. Exp. Botany 42(242): 1117-1122.

4. Lang, A. and M.R. Thorpe. 1989. Xylem, phloem and transpiration flows in a grape: application of a technique for measuring the volume of attached fruits to high resolution using Archimedes' prinCiple. J. Exp. Botany 40(219): 1069-1078.

5. Rosenquist, J.K. and J.C. Morrison. 1989. Some factors affecting cuticle and wax accumulation on grape berries. Am. J. Enol. Vitic. 40(4):241-244.

67 YEASTS IN WINEMAKING

Roland Riesen Department of Horticulture The Ohio State University/OARDC, Wooster, OH 44691

HISTORY

1697 Fermentation theory by Stahl "An object which is subject to an intense inner movement can tear another object which is still quiet into the same dramatic movement".

1632-1723 Antonius van Leeuwenhoek: development of the first microscope (magnification: 100 to 150 fold).

1789: Lavoisier: proved ethanol and C02 as main products of the alcoholic fermentation.

1803 First consideration ofyeast as cause ofthe alcoholic fermentation (Erxleben, De La Tour, Schwann, Kutzing).

1810 Gay-Lussac: establishment of the complete fermentation equation.

1860 Louis Pasteur: "Ia fermentation est Ia vie sans air" (fermentation is life without air) 1866 Louis Pasteur: "etudes sur levin" (studies on wine) 1876 Louis Pasteur: "etudes sur Ia bierre" (studies on beer)

1880's Christian Emil Hansen: use of pure yeast cultures in brewing

1894 Julius Wortmann (Geisenheim): Foundation ofthe first pure culture collection for wine yeast.

1897 Eduard Buchner (Wurzburg): Nobel Prize for the discovery of fermentation enzymes.

68 (.... D G~ .······-. .: ... . ·:: . ... . C·0:. .

B H•

o. fh[I 3· . E•• • ..... c F =-·.....

Figure 1. Leeuwenhoek's drawings ofbacteria, published in 1684. Even from these crude drawings we can recognize several kinds of common bacteria. Those lettered A. C, F, and G are rod-shaped; E, spherical or coccus-shaped; H, coccus-shaped bacteria in packets. (Leeuwenhoek, A van. 1684. Phil. Trans. Roy. Soc. London 14:568).

69 ~ ~ (b)

·' ..

..· / ,~....

(~) (d)

Figure 2. The yeast cell as it seemed to different observers. The great incre....e m our understanding of cell structure came with improvement in our microscopes. (a) Leeuwenhoek's drawing of yeast, dating from 1694. Note the complete absence of any cellular detail. (From Leeuwenhoek, A van. 1694. Ondervindingen en Beschouwingen der Onsigtbar Geshapene Waarheden, 2nd ed. p. 45. Delft.) (b) Pasteur's drawings ofyeast, made in 1860, showing the budding process by which yeasts grow. The contrast of the outer cell wall and inner cytoplasm is distinct. The large objects in the cytoplasm are vacuoles. (From Pasteur, L. 1860. Ann. Chim, Phys. 58:323). (c) Drawing ofthe idea of a yeast cell in 1910. The greater detail inside the cell derives partly from improved microscopy and partly from the use of dyes that increase contrast and stain particular structures. However, some ofthe labeled structures are probably artifacts. (From Wager, H. and A Peniston. 1910. Ann. Bot. 24:45) (d) Photograph of a yeast cell as seen with the electron microscope. The cell is first treated with chemicals that preserve the structure and stain particular components. Magnification 31,200x. (From Conti, S.F. and T.D. Brock. 1965. J. Bacteriol. 90:524).

70 DEFINITION, CLASSIFICATION

Yeasts belong to the group ofthe fungi:

saccharo sugar (greek: saccharon) myces fungus (greek: myketes) cerevisiae brewing (latin)

Saccharomyces cerevisiae: "brewer's sugar fungus"

Since the discovery of the yeasts the scientists have struggled for a universal definition but haven't completely succeeded until today because they consist of many diverse organisms. As a consequence the definition rather describes the properties and non-properties of yeasts based on the following criteria: appearance reproduction mode nutritional characteristics fermentation characteristics

shape: round, oval, ellipsoidal or cylindrical (fig. 3 and 4). The shape changes with the nutritional composition of the growth medium.

size: variable from small to medium to large: small: (2J..l-7J..l) x (4J..l-l5J..l) large: (4J..l-lOJ..l) x {7J..l-2lJ..l) growth: single cells, pairs, occasionally short chains. reproduction: 2 different reproduction modes (fig. 5): - sexual formation of ascospores, charact. process: cell meioses (fig. 6) - asexual (vegetative) no formation of ascospores, charact. process: budding (fig. 7).

The vegetative reproduction mode (budding) is common to All yeasts whereas the sexual reproduction mode (cell meioses) has not (yet) been discovered for all yeasts, but may still exist and can, therefore, not be excluded! The difference in observed reproduction modes is used to classify the yeasts.

71 Fig. 3. Saccharomyces cerevisiae. Young, budding pure culture after 3 days in grape must (Lemperle and 1982).

Fig. 4. Saccharomyces cerevisiae. Resting yeast cells in young wi after fermentation. (Lemperle and Kerner 1982).

72 r-') ® ~~(!)J~ .A.sexual G!) @' reproduction ~

~ \!1;; Haploiti \!' Mating of opposite types Germination

~ c. •,!) ® Ascospores ®® l Cell fusion ~

@@@ Nuclear fusion ~~~ Ascus ~• .A.sexual reproduction Meiosis

Dipioiti

Fig. 5. Life cycle of a typical yeast. Saccharomyces cerevisiae.

73 Fig. 6. Schizosaccharomyces pombe. Multiplication by segmentation (magnification 1500-fold, phase contrast, Luthi et al. 1982) A begin segmentation B. before end of segmentation

74 Fig. 7 Vegetative cell cycle of Saccharomyces cerevisiae (Dittrich 1987).

75 Fig. 8. Saccharomyces cerevisiae. SEM picture ofbudding yeast cells. (P. Gruss et al. 1984).

76 CLASSIFICATION OF YEASTS

Yeast-like fungi

1. Fungi with known ascospore formation ("perfect form")

1. Family 1. subfamily

L genus: Schizosoccharomyces 2. subfamily

L genus: Saccharomycodes, Hanseniaspora

4. subfamily

L genus: Saccharomyces, Zygosaccharomyces, Pichia, Hansenula, Dekkera

2. Fungi with unknown ascospore formation ("imperfect form")

1.

genus: Brettanomyces, Candida, Kloeckera

77 OCCURRENCE- WILD YEASTS

Yeasts occur everywhere where there are sugar solutions: in saps of plants and trees, in the nectar of flowers and on :fruits such as grapes. Intact grapes are protected by a wax layer which prevents the yeasts from penetrating. They still occur on the surface of the wax layer, but only in low numbers and they can't multiply because they lack the nutrients. Once they have access to the nutrients, e.g., through cracks in the skin, they multiply rapidly and start the fermentation process while the berries are still on the vine. In addition the sugary solution attracts other microorganisms and insects such as ~asps and :fruit flies which are the prime carriers to spread the yeasts and the other microorganisms further. The effectiveness and persistence of this yeast transfer has been demonstrated by the isolation of living and reproducing yeast cells not only from the legs and tongs of :fruit flies, but also from their intestines.- ..

DOGMA : Saccharomyces cerevisiae = King of nature

A dogma prevails that the wine yeast par· excellence, Saccharomyces, is ubiquitous in nature, that actually it has a definite preference for vineyard and orchard soils, that it survives the winter in the soil, resurges in spring, allowing it to dominate its empire again, year after year. Moreover, that each microclimate hosts a specific yeast flora naturally selected and optimized for the task of fermenting the must of that specific grape variety grown in that specific environment.

TRUTH : Saccharomyces cerevisiae = the domesticated animal

The yeasts in nature - the so-called "wild yeasts" - are the same all over the world, they differ only in their relative proportions which are strongly dependent on the climatic conditions. As a consequence the composition of the wild yeast flora at a specific location changes yearly with the yearly meteorological differences leading to an ever-changing yeast flora in the must and, if relied solely on these yeasts for fermentation, an unpredictable course of the alcoholic fermentation. The wild yeast population is dominated by apiculate yeasts, film yeasts and non-fermenting yeasts with no or only a low fermentation capacity and with a dominating aerobic metabolism. The wine yeast Saccharomyces is practically absent independent of the ripening period. The only location where Saccharomyces dominates is the winery itsel£1 Martini et al. ( 1) investigated the degree of dominance by using labelled yeast in a newly established winery. After two years ofwinemaking all the surfaces of the winery were colonized by the labelled yeast. In the third year when no starter culture was used the labelled yeast dominated the fermentation rapidly, inhibiting the growth of the wild yeasts from the grapes.

78 The yeast ecology of various natural and artificial habitats associated with the winemaking process can be summarized as follows (2):

Habitat Specific microflora

1. Where grape must is produced (soil, species with predominantly oxidative air, fresh water) metabolism; wine yeasts are absent!

2. Sugary fruits in general Metschnikowia pulcherrima and Kloeckera apiculata dominate; non-fermenting species are always present; Saccharomyces cerevisiae is essentially absent.

3. Grape berries in particular Metschnikowia pulcherrima and Kloeckera apiculata; Saccharomyces cerevisiae is practically absent independent of the ripening period.

4. Harvest of grapes (hands of pickers, Metschnikowia pulcherrima and Kloeckera buckets, crates) apiculata predominate.

5. Fermenting must Kloeckera apiculata initiates the fermentation with, hopefully, rapid substitution by Saccharomyces cerevisiae which terminates the process. Torulaspora delbrueckii can partially take over the fermentation in some cases.

6. Winery (walls, floors, equipment) Saccharomyces cerevisiae predominates; minor presence of film-forming yeasts.

Table 1 Yeast ecology ofvarious natural and artificial habitats associated with the winemaking process; Peynaud, E.: Am. J. Vitic. Enol. 10:69-77 (1959).

79 "WILD YEASTS" - SPONTANEOUS FERMENTATION

Composition: 10-30 genera, 50-150 species:

main representatives fermentation characteristics

Kloeckera apiculata weak fermenter Torulopsis weak fermenter

Metschnikowia pulcherrima film-forming yeast (aerobic) Candida film-forming yeast (aerobic) Pichia membranaefaciens film-forming yeast (aerobic) Hansenula anomala film-forming yeast (aerobic)

Rhodotorula non-fermenter Aureobasidium non-fermenter

Several studies have shown that the group with an aerobic metabolism varies most with the climatic conditions, and that it grows particularly well in rainy and cool season.

Cell number: unpredictable!

The cell number varies anywhere from a few cells/berry to several 100 million cells/berry, depending on the climatic conditions and the soundness of the berries. Generally, it can be assumed that in a fresh pressed must 0.1% or less of the yeasts present is the wine yeast Saccharomyces.

Natural selection by the specific must parameters: high acid content (low pH): most yeasts survive, most bacteria die (exception: acetic acid bacteria, some lactic acid bacteria). high sugar cootent: fermenting yeasts survive, non-fermenters do not.

502 addition (voluntary): most bacteria and wild yeasts have no or a very low tolerance towards S02.

80 Fermentation characteristics of a spontaneous fermentation:

initiation : apiculata yeasts!

genera: Kloeckera (imperfect form) Hanseniaspora (perfect form) Kloeckera/Hanseniaspora are the same yeast representing the two different reproduction modes.

size: small (1.5~-5~) x (2~-11 ~) smaller than Saccharomyces, sometimes as small as bacteria ("flying yeasts").

shape: apiculate, sometimes lemon-shaped, sometimes sausage-like (fig. 9). The shape depends on the nutritional composition of the growth medium and on the age of the cells.

growth: single cells or occasionally short chains. The reproduction cycle is about twice as fast as that of Saccharomyces.

metabolism: aerobic! fermentation capacity: low (pure cultures: 5-6% alcohol). alcohol tolerance: low (max. tolerance around 4%) antagonism: possibly antagonistic (in large numbers) and stimulatory (in low numbers) towards Saccharomyces.

A typical spontaneous fermentation is not a pure culture fermentation, but is characterized by a succession of genera, species and strains. Generally it is initiated by apiculate yeasts and finished offby Saccharomyces.

81 Fig. 9 Kloeckera apiculata. Young, budding pure culture in grape must (magnification 800- fold. phase contrast. Lemperle and Kerner 1982).

82 Sensory characteristics of a spontaneous fermentation:

"more complex, softer, rounder, fuller wines" facts: increased formation of:

-acetic acid 0.5-1.2 g/1 -ethyl acetate in small amounts: may add complexity, but distracts from varietal character. in higher amounts: solvent, airplane glue, nailpolish remover, typical compound formed during carbonic maceration. -glycerol odorless, colorless, slightly sweet tasting liquid (body). -higher alcohols strong, pungent, fusel. -2-phenylethanol rose, honey, fragrant, floral. -802

decreased formation:

-acetaldehyde pungent, oxidative, , sharp, ethereal, coffee (on dilution).

Advantages of spontaneous vs selected pure culture fermentation: advantage of a spontaneous fermentation:

"more complex, softer, rounder, fuller wine" advantages of a selected pure culture fermentation:

-rapid onset of fermentation -even, predictable and complete fermentation -predictable rate of sugar-to-alcohol conversion

-improved alcohol and 802-tolerance -improved adaptation to low temperatures

-controlled production of 802, acetic acid, acetaldehyde, ethyl acetate, higher alcohols -controlled production of desirable by-products -controlled production of undesirable by-products -choice of specific yeast attributes (low foaming, glycolytic activities, flocculation properties)

83 ALCOHOLIC FERMENTATION

1810: Gay-Lussac: alcoholic fermentation equation:

sugar alcohol gas glucose, fructose ethanol carbon dioxide

sugars= carbohydrates= saccharides (mono-, di-, tri-, oligo-, polysaccharides)

com sugar, dextrose glucose (monosaccharid) fruit sugar fructose (monosaccharid) beet sugar, saccharose, sucrose, invert sugar glucose-fructose (disaccharid) lactose, milk sugar glucose-galactose ( disaccharid) theoretical yield of ethanol 51.1% of sugar (w/w) practical yield of ethanol - 47% (w/w) yield loss : evaporation yeast growth (1% of sugar) formation ofby-products

Formation ofby-products

Yeasts can metabolize sugar in 2 different modes:

-anaerobic alcoholic fermentation, low energy yielding, reductive process.

-aerobic respiration, high energy yielding, oxidative process.

The types and amounts of by-products formed depend on which process is given priority at any chosen moment during the alcoholic fermentation. The sugar metabolism of yeasts is summarized in Figure 10.

84 acetic acid

respiration (C02, li20) fatty acids fats

esters higher alcohols nino acids .------.., I • acety1-CoA. , L------J acetic acid

aerobic oxidation glucose fructose ~ I 2 pyruvate I c=) I 2 acetaldehyde I + 2 co~

anaerobic /',_' 2 ethanol glycerol lactic acid

Fig. 10 Sugar metabolism of yeast.

85 pyruvate. acetaldehyde

significance: binding partners for S02, sherry-type, oxidative aroma (acetaldehyde)

pyruvate increased levels with: -high fermentation temperatures -botrytis infection -pasteurization acetaldehyde: increased levels with: -extremely slow fermentation -addition of so2 during fermentation -stopped fermentation (partially fermented sweet reserve, Fig. 11 ). -nutrient deficiencies

range in wine: 6-190 mg/1 (yeast dependent) aroma threshold in wine: 100-125 mg/1 descriptors: green leaves, pungent, sharp, sherry, oxidative, coffee (on dilution)

(gil) (mg/1)

•.80 200

70 175 alcohol \ _, 0 0 c60 150 .2.. ·x/_. ~so 125 ~ <: 0 ~40 100 I. ·, 30 75 ~ ·,.:cetald~::::·-- 20 • ...... ·-· • 1___.. ·-·-·-· • • • • .- s TO 15 20 Zeol (Taget

Fig . 11. Formation of acetaldehyde during fermentation.

86 acetic acid main mechanism of formation: enzymatic oxidation by acetaldehyde dehydrogenase

oxidation acetaldehyde ---.> acetic acid

Saccharomyces cerevisiae: 0.02 - 0.5 gil "wild" yeasts 1 gil or more (Kloeckera apiculata, Hansenula anomala, Candida krusei) the reaction is directly correlated to the sugar metabolism: the end of the alcoholic fermentation means the end of acetic acid formation by this mechanism. glycerol characterization: colorless, odorless, slightly sweet tasting, viscous liquid (body, mouthfeel of wine) main mechanism of formation: shunt reaction during the alcoholic fermentation: the formation is

strong, when the main substrate for NADH2 - acetalydehyde - is not readily available (beginning of fermentation, vigorous fermentation).

other source: botrytis!! rangem wme: 8-10% of ethanol content ("wild" yeast> Saccharomyces) lactic acid main mechanisms of formation: pyruvate > lactic acid lactic acid dehydrogenase, LDH the reaction is seldom ( 100-200 mglllactic acid) because most yeasts lack the adequate enzyme system.

87 "fermentation aroma"

More than 90% of the "fermentation aroma" is made up of 14 components belonging to 4 classes:

higher alcohols acetate esters of higher alcohols fatty acids ethyl esters of fatty acids

The "fermentation aroma' varies considerably with the yeast and with the fermentation conditions, but not so much with the grape variety. Most affected by the fermentation conditions are the higher alcohols and the acetate esters.

higher alcohols (fusel alcohols)

occurrence: quantitatively the largest group of flavor compounds in alcoholic beverages

descriptors: strong, pungent, fusel main components: aroma concentration threshold in in wine (mg/1) wine (mg/1)

3-methyl butanol 60-150 60 (isoamyl alcohol) 2-methyl butanol 10- 30 -30 (act.amyl alcohol) 2-methyl propanol 20- 80 200 (isobutyl alcohol) !-propanol 20- 60 750 significance: they are more toxic than ethanol and can influence membrane processes in certain cell types. they increase the duration of ethanol in the body and hinder its metabolism. they play a crucial role in the "hangover". main mechanisms offormation: direct sugar metabolites (fig. 10,12).

88 sugar

Amino acids I ~ fermentation (Pyruvatl proteins r Acetyl-GoA-activated fatty acids Amino acids I J TCA cyc1e fatty acids Jt fats (trans)amination NH£ ~r t o a c i d s

ethanol higher ester alcohols

Fig. 12. Intracellular metabolism of Saccharomyces cerevisiae: formation of ethanoL higher alcohols and esters.

factors influencing the level of higher alcohols:

-sugar content of must: the higher the sugar content the higher the level of higher alcohols.

-free amino acid content of must: amino acid deficiencies lead to an increased formation ofhigher alcohols (see fig. 12).

-juice clarity: particulate matter is the most important parameter influencing the level of higher alcohols.

1) the larger the particles the greater the production of higher alcohols. 2) at a specific particle size: the more particles the greater the production of higher alcohols.

-fermentation temperature: the higher the temperature the greater the production of higher alcohols.

Yeast surface of 108 cells/ml: 16.5 acres/1000 gal

•

89 occurrence: numerically the largest group of flavor compounds in alcoholic beverages. descriptors: fruity, flowery, sweetish, apple-like, banana-like, soapy range in wine: 200-400 mg/1 (total ester content) main mechanism of formation: enzymatically within the cell factors influencing the level of esters:

-yeast species, strain -amount of yeast: increased amount of yeast increases the ester content -sugar content of must: increased sugar content increases the ester content -juice clarity: increased clarity increases the ester content -fermentation conditions (temperature, aeration): no general statement for the esters as a group is possible; the optimum condition for the ester formation varies with each individual ester. classification: 1) ethyl esters of fatty acids: fatty acid + ethanol --> ethyl ester

2) acetate esters of higher alcohols: acetic acid + higher alcohols --> acetate ester

•

90 FILM YEASTS

Classification

-Hansenula : 30 species (H. anomala) strong ester formation, 2-phenylethanol

-Pichia 56 species (P. membranaefaciens, P. vini, P. farinosa) aldehydic

-Candida 196 species, imperfect form ofPichia and Hansenula (C. stellata, C. pulcherrima, C. vini) higher alcohols, 2-phenylethanol, ethyl acetate

Occurrence nature: film yeasts are a part of the "wild" yeast population and help initiate the fermentation. Their fermentation strength varies widely among the genera and species, but they are altogether weaker fermenters than Saccharomyces. They usually die off quickly after the beginning of the fermentation because they are strictly aerobic - and the oxygen content of the must is depleted very rapidly. After the fermentation they can grow again, but only if there is oxygen available. They don't grow in the wine, but on the wine surface because of the availability of oxygen. wmery: everywhere where there are nutrients and oxygen available (partially filled tanks, open containers, not frequently enough topped barrels, equipment, floors which are not thoroughly cleaned after use).

Appearance

Depending on the degree of infection: ugly looking and somewhat distractive smelling and tasting, continuous, remarkably, stable, velvety, soft, white, creamy, chalky, wrinkly, dusty-floury film floating on the wine surface and growing in thickness ..... (fig. 13,14).

91 Fig. 13. Film yeast. Old, heavy pellicle on surface of grape juice (magnification 6-fold, bright­ field illumination, Luthi et al. 1981 ).

Fig. 14. Film yeast. Old, heavy pellicle on surface of grape juice (magnification 6-fold, bright­ field illumination, Luthi et al. 1981 ).

92 Characterization

-alcohol tolerance : variable, generally low

-S02 tolerance : variable

-descriptors estery, artificially estery, aldehydic, lack offiuit and varietal character, oxidized, thin, empty, naked, ......

Metabolism

-sugars, anaerobically (fermentation) occasionally

-sugars, aerobically (respiration) strong

-ethanol, aerobically (oxidation) main energy source!

- organic acids, aerobically yes

esters higher alcohols

1\ I ox ox ethanol + 0 2 --> acetaldehyde --> act acetic acid

I respiration V (TCA cycle)

the composition of a wine, affected by Pichia farinosa, was changed in the following way (3,4):

initial after 3 months

ethanol 70.8 gil 20.5 gil malic acid 6.8 gil 3.4 gil glycerol 6.9 gil 1.3 gil (6 months)

93 Fig. 15. Pichia membranaefaciens Fig. 16. Pichia membraneaefacines. After 3 days in malt extract After 10 days in potato agar (Lodder 1970) . (Lodder 1970)

o· <2:) ~

Fig. 17. Dekkera bruxellensis. Pure culture after 5 days in malt extract (Lodder 1970).

94 APICULATE YEASTS

Classification

-Kloeckera!Hanseniaspora -Saccharomyces -Zygosaccharomyces -Brettanomyces/Dekkera

Saccharomycodes

-nickname sulfur yeast! -species Saccharomycodes ludwigii -occurrence in spontaneous fermentations of fruit-, berry- and grape juices -growth large single cells form sediments, colonies -significance very, very high S02 tolerance; a strain isolated in Spain was able to ferment grape must with 500 ppm free and 2000 ppm total so2! -defects sediments; the sediments are not easy to detect because even during the fermentation they settle at the bottom and the wine above is clear. No production of off-aromas.

Zygosaccharomyces

-nickname : sulfur yeast! -species Zygosaccharomyces bailii (haploid form of Saccharomyces) -occurrence in fruit concentrates -growth large, compact lumps -significance high osmotolerance high so2 tolerance high sorbate- and benzoate resistance -defects sediments

95 Brettanomyces/Dekkera

The myth and range of controversy about "Brett" is illustrated best by the two following quotes:

journalist : "one prominent wine writer, in particular, reportedly has such a strong preference for Brett flavors that he consistently ranks infected wines higher than uninfected ones. As a result, some wineries are deliberately producing wines that are at least slightly infected, possibly to improve their ratings."

microbiologist: "Brett is the chief microbial concern in most wineries, it is~ spoilage yeast par excellence.

-classification: Brettanomyces/Dekk:era are the same yeast but with a different reproduction mode.

Brettanomyces :imperfect form (7 species) Dekk:era : perfect form (2 species)

-shape: range from ogival to ovoidal, strong polymorphism (Fig. 17).

-size: usually small ("flying yeast") (l.5J.1-3.5J.1) x variable length

-growth: Very versatile - not a sweet wine problem! Brett has been isolated from red wines, dry white wines with 15% alcohol, , sparkling wines. Sometimes the incomplete separation of the daughter cells from the mother cell leads to the formation of a film.

fermentation strength weak and slow alcohol tolerance high (14-15%)

S02-resistance frequently resistant to normal so2 levels sorbate resistance high actidione resistance very strong (up to 100 ppm!)

-ongm: winery!! Dirty crush equipment, drains, pools of grape juice left over from cleaning, cooperage. The concept that Brett enters the winery on grapes cannot be supported by extensive research all over the world: Brett lives in the winery!!

96 Once Brett is in the winery, it is very difficult to get rid of The best control can be achieved by preventive measures:

-control : prevention, by I) sanitation, particularly important is the elimination of any food sources (need of external vitamins for growth). 2) maintenance of adequate so2 levels (deterrent reducing the growth without complete elimination ofthe organism) 3) segregation of infested wine lots 4) maintenance of cool cellar temperatures

-descriptors: distinguished fruity, apple- or cider-like, aldehydic, sharp, sour, burnt beans, ammonia, mouse dropping, horse sweat, pungent scent of barnyard animals, underlying notes of spicy, clove-like, smoky, woody, phenolic - and a bitter, metallic aftertaste).

-rationalization of aroma descriptors: the formation of the by-products responsible for the various growth descriptors depends largely on the fermentation conditions, particularly on the degree of oxygen present, since the Brett fermentation is stimulated by oxygen (Custer effect; semi-aerobic or aerobic fermentation).

I) fruity, apple- or cider-like, aldehydic: high activity of esterification enzymes: increased formation of ethyl acetate, isoamyl acetate, 2-phenethyl acetate, ethyl esters of medium chain fatty acids (Cs-Ct4).

2) sharp, sour, pungent: increased formation of -acetic acid: up to 2-3 g/1 -short chain fatty acids: rancid, sweaty, cheesy, penetrating, putrid.

-medium chain fatty acids (C8-C14): inhibition of Saccharomyces

3) burnt beans, ammonia, barnyard animals, mousiness: lysine + ethanol > 2-acetyl-I, 4,5, 6-tetrahydropyridine + 2-acetyl-3 ,4 ,5,6-tetrahydropyridine

4) strongly spicy and clove-like: metabolism of ferulic acid to 4-ethylguaiacol

5) woody, smoky, phenolic: metabolism of p-coumaric acid to 4-ethyl phenol

97 THE MYTH OF BRETT•...

1989 Shows Its Greatness Again

..••BETWEEN DREAM, FACT AND UNCHANGEABLE REALITY:

" .•. we decided that a little Brett adds warmth and complexity. As the wine develops, the barnyard aromas disappear, leaving earthy and cedar notes. ( .•• ). We want to control Brett, but not get rid of it entirely."

98 BACTERIAL CONSIDERATIONS IN WINEMAKING

Christian E. Syberg, Winemaker Meier's Wine Cellars, Inc., Cincinnati, OH

ABSTRACT

This paper reviews some of the literature from the point of view of source, impact and strategies for their control.

INTRODUCTION

Much of the literature is suggesting a word of caution to believe that our efforts of controlling bacterial growth at all stages ofwinemaking is adequate. Unless special attention and management of all stages of grape harvest, fermentation, bottling and wine storage is exercised, there will be increasing incidents where the wine will be modified as a result of bacterial activity.

Vinification is a complex interaction between several groups of organisms with the potential of destruction as well as creative joy. Given the right circumstances and proper/improper caretaking chemical alterations will take place in all its various degrees of expression.

The role of bacteria in winemaking can be found among relatively few species, namely, Acetobacter, Gluconobacter in one group, Lactic acid bacteria in a second and the little known Bacillus in a third group. Generally, wine is a relatively hostile environment for most bacteria, so by comparison with other foods we are relatively lucky.

Juice, in general, is far more susceptible to bacterial growth than wine, emphasizing the importance of well established harvest criteria and processing methods.

Acetobacter and Gluconobacter

The most recent taxonomic classification of these genera places them in the same family asAcetobacteraceae (Bergeys Manual ofDeterminative Microbiology, 9th edition). This recognizes their ability to oxidize ethanol to acetic acid. The family consists of two genera, Acetobacter and Gluconobacter. Gluconobacter represented by a single species, G. oxydans, whereas, Acetobacter by four species, A. aceti, A. pasteurianus, A. liquifaciens, and A. hansenii.

99 Acetic acid bacteria (ABB) are gram-negative rods (gram-variable in older culture) often occurring in pairs or chains. Their size varies from 0.6-0.9 J.lm by 1 to 3 J.lm. G/uconobacter may exhibit polar flagellation.

Both Acetobacter and Gluconobacter exhibit respiratory (aerobic) metabolism and, therefore, you will find them normally on the surface of the wine as a translucent, adhesive film. The formation of surface film or pellicles may cause the wine to become hazy or cloudy. However, there organisms will survive at very low oxygen tension (semianaerobic) and as a result can be found in wines under most conditions.

Their origin stems mainly from grapes/must and winery equipment. The main isolates are Acetobacter aceti, Acetobacter pasteurianus and Gluconobacter oxydans. The population varies with the condition of the grape/must from 100 cellslmL to 106 cellslmL. On sound grapes the AAB are dominated by Gluconobacter oxydans, whereas, on damaged/spoiled material the population shifts to the Acetobacter species aceti and pasteurianus. Though ABB are considered to be obligate aerobes, vigorous carbon dioxide production during fermentation alone is not sufficient to control their growth. Studies have indicated that the proportion of AAB/yeast in must will determine the growth kinetics of AAB during the alcoholic fermentation. It should be noted that acetic acid can be produced during the alcoholic fermentation if the initial population of AAB is high enough (100-106 cellslmL). Under normal circumstances there is little tendency to grow during fermentation. At the end of the fermentation there are generally less than 100 cellslmL. After fermentation, any transfer of the wine should be regarded as a point of agitation/aeration, which as a result could encourage growth of AAB significantly.

Factors affecting survival and growth of ABB are pH, temperature, ethanol concentration, sulfur dioxide and dissolved oxygen:

pH: In a juice/vinificationlwine environment, though pH is not at optimum growth at pH 3.0 to 3.5 is well documented. The capacity of survival and growth decreases as pH decreases. In all likelihood the nutrient requirements increase with a decrease in pH. The sensitivity to alcohol increases as pH decreases and lastly, pH will effect their ability to metabolize glucose, lactate and ethanol.

Temperature: Though optimum temperatures reported are in the range of20°C to 35°C, the minimum temperature where growth ceases has not been established. It has been shown that Acetobacter aceti shows weak growth at 10°C, suggesting that low cellar temperatures will not prevent growth of AAB completely.

Ethanol: The metabolism of ethanol is the distinguishing feature of AAB and is well documented as the basis of wine spoilage. There is limited information regarding ethanol tolerance. Isolation of Acetobacter from wines clearly suggests that these organisms can grow in the range of 10% to 15% ethanol.

100 Sulfur Dioxide: Studies conducted on the growth of AAB indicate that the concentrations used during normal winemaking practices are not sufficient to control their growth. The literature reports a wide range of sulfur dioxide tolerance without any reference to the molecular portion of free sulfur dioxide which is pH dependent. Inhibition of most AAB can be achieved with a molecular sulfur dioxide of0.8 mg/L.

Oxygen: As obligate aerobes, ABB use oxygen as electron acceptor. It is well documented that growth in wine is associated with exposure to air which can result in the development of surface flora and wine hazing. Recent studies indicate their ability to grow under reduced oxygen conditions and survive under anaerobic conditions, emphasizing the importance of avoiding aeration (pumping and transfers). Wooden cooperage permits the transfer of 3 0 mg!L/year of oxygen which is sufficient for survival of low populations of AAB.

SIGNIFICANCE IN WINEMAKING

Carbohydrates: AAB lack a functional EMP pathway and, therefore, cannot metabolize hexoses via the EMP pathway. However, they will metabolize hexoses and pentoses after phosphorylation via HMP pathway to acetic and lactic acid.

1: HMP

phosphorylated hexoses or pentoses -->acetic acid+ lactic acid (Acetobacter spp + Gluconobacter oxydans)

--> carbon dioxide + water ( overoxidation by Acetobacter aceti via TCA cycle)

2: hexose --> gluconic acid --> keto gluconic acid (direct oxidation)

The utilization of glucose via HMP pathway versus oxidation to gluconic and ketogluconic acid via pathway 2 depends upon pH and glucose concentration. At pH <3.5 and glucose concentration >5-15 mM the HMP is inhibited or repressed, resulting in the accumulation of gluconic acid. Further oxidation of gluconic acid into ketogluconic acid occurs only at pH >3.5 and glucose concentration <10mM.

Growth of AAB on grapes and in must can lead to high concentrations of gluconic and ketogluconic acid. The production of these substances by this mechanism is more important than their production by Botrytis cinerea. Gluconobacter is capable of strong growth on infected berries and subsequent oxidation of glucose to gluconic and ketogluconic acid. Acetobacter are weaker in the hexose utilization and tend to produce less gluconic acid.

Other by-products of the carbohydrate metabolism are dihydroxyacetone; 2,3-butanediol; acetoin; extracellular polysaccharides: cellulose, dextran, levans (can affect filtration).

Glycerol: is formed during fermentation by the yeast and is important to winemaking for its

101 contribution to mouth feel and slight sweetness. Normal concentrations are in the range of 3-14 giL. In grapes infected with Botrytis cinerea, the amount of glycerol is significantly increased: up to 20 giL as a consequence of the metabolism of sugars and acids. The . significance of glycerol utilization by AAB can be attributed to Gluconobacter oxydans and Acetobacter aceti. Both organisms can oxidize glycerol to dihydroxyacetone.

The conversion is dependent on pH, oxygen and alcohol concentration. At pH 3. 0-4.0 there is substantial activity, the optimum pH is 5.0. Ethanol at 5% is inhibitory to the reaction. The formation of dihydroxyacetone is likely to have some effect on the sensory wine quality: sweet aroma and cooling taste. It can be produced in grape must as high as 260 mg!L with a carryover of 133 mg!L into wine. It is not known if the reaction occurs also in wine.

Ethanol: The winemaker's familiarity with AAB cannot be more simply stated than with his awareness of ethanol being oxidized to acetic acid. It is a well-known process in vinegar production.

Ethanol-> acetaldehyde-> acetic acid (Acetobacter, Gluconobacter) -> carbon dioxide+ water (overoxidation by Acetabacter spp. via TCA cycle)

Optimum pH for this reaction is 5.0. In wine there is substantial activity at pH 3.0-4.0. High oxygen concentration favors the reaction, though in anaerobic environments oxygen can be replaced as electron acceptor by quinones, permitting near anaerobic conditions. Low oxygen concentrations do, however, favor the accumulation of acetaldehyde.

The spoiled character associated with volatile acidity is the result of the formation of ethyl acetate. This ester is perceived as the odor of fingernail-polish remover. It has been suggested that the concentration of ethyl acetate should replace volatile acidity, as a measure of the soundness of a wine. It is possible to have a wine which is sensorially sound, but too high in volatile acidity, as it is possible to have a wine low in volatile acidity, but poor quality as a result of a high ethyl acetate concentration. Aroma threshold for ethyl acetate is 160-180 mg!L. (Flavor threshold: 100-125 mg!L, normal wine concentrations 20-200 mg!L.) Greater than 500 mg!L is considered detrimental to wine. AAB can produce up to 160 mg!L.

Organic acids: Considerable changes in acidity can be caused by AAB in that some A.cetobacter can completely oxidize acids such as lactic, pyruvic and acetic acids, via the TCA cycle, to carbon dioxide and water. Other acids such as malic, succinic, citric, and fumaric acid can also be oxidized, but this is very much dependent on the particular organism. Not all acids will be oxidized by the same species. As previously mentioned, Gluconobacter oxydans does not have a functioning TCA cycle and is, therefore, not able to degrade acids.

CONTROL OF ACETIC ACID BACTERIA

The assumption that the absence of oxygen will control this group is questionable. Evidence is suggesting that they can survive in wine under low oxygen conditions, as can be

102 found in wooden barrels, or tanks with ullage. Other factors such as pH and alcohol also affect growth and survival. At a pH ofless than 3.2 and an alcohol content over 13% there is little growth. The maximum tolerable alcohol content reported is 14 to 15%.

Sulfur dioxide at 0.8 mg/L (molecular) is usually adequate to inhibit most acetic acid bacteria. It should be pointed out that stratification of S02 as well as alcohol does occur in tanks; thus reminding us that the surface ofwines may not be analytically representative. Leaky tanks, pools of wine, rough surfaces which are difficult to clean, dirty or leaking barrels and partially filled tanks are all good places for ABB growth. Not enough can be said about proper sanitation and storage. -

The condition ofthe grapes at the time of harvest and grape handling are probably the most important factors which contribute to the proliferation of acetic acid bacteria if not addressed properly.

LACTIC ACID BACTERIA

The significance ofthis group ofbacteria stems from their ability to reduce acidity and increase the pH. Lactic acid bacteria (LAB) convert L-malic acid to L-lactic acid and carbon dioxide in an enzymatic process referred to as malolactic fermentation (MLF), even though it is not a true fermentation. Several by-products, which are dependent on the organism involved, are produced. They may have an impact on the sensory properties of the wine and its complexity.

The origin of lactic acid bacteria has been attributed to their presence on grapes and vine leaves where they have been enumerated at various concentrations depending on the maturity and condition of the berries and the vine. Laboratory prepared wine (no contact with winery equipment, Kunkee et al.) can undergo MLF indicating grapes are the source of LAB. However, winery equipment such as tanks, pumps, valves, wooden barrels and bottling equipment are also implicated as sources. Studies have shown that grapes and winery equipment are the principle sources oflactic acid bacteria. Several genera of Lactobacillus, Leuconostoc and Pediococcus can occur in wine. All are, therefore, alcohol tolerant. Leuconostoc oenos is considered the preferable genus. It is best able to tolerate low pH conditions. Several strains ofLeuconostoc oenos are commercially available as starter cultures.

The harsh physio-chemical and low nutritional properties of wine inhibit all but a few species ofLAB. Must soon after crushing generally contains LAB at levels of 100-1 0000 cells/mL. The major species present at this stage are Lactobacillus plantarum, Lactobacillus casei and Leuconostoc mesenteroides, Leuconostoc oenos and Pediococcus cereviseae. They generally do not multiply much and die off during fermentation. On odd occasions there can be a slight proliferation of some species (L. plantarum). By the end ofthe fermentation, the population ofLAB has declined to but a few cells/mL (not detectable by plating on agar media).

103 After a lag phase, which depends on wine properties, the few surviving cells begin to multiply and eventually conduct MLF. This phase is characterized by vigorous growth and a population of 106 to 107 cells/mL. It is almost invariably Leuconostoc oenos which develops at this stage even though in wines with high pH; ie., 3.5 to 4.0, Pediococcus or Lactobacillus can grow as well. The subsequent fate of LAB during cellar storage depends on the way the wine is handled after completion of the MLF.

FACTORS WIDCH AFFECT SURVIVAL AND GROWTH OF LAB IN WINE

pH: Wine pH is one of the most important parameters affecting the behavior ofLAB. pH influences: a) lag phase leading up to MLF; b) rate of growth and duration ofMLF; c) species that grow in wine; d) metabolic behavior of the species that grow; e) survival ofLAB. MLF is completed faster as the pH increases above 3.0. At the lower limit, it can take approximately 165 days versus 14 days at pH 3.9. Good averages are 6 weeks above pH 3.5 and 12 weeks or longer for pH below 3.5. Further, the selective effect of pH is such that at pH below 3.5 growth of Pediococcus and Lactobacillus spp. does generally not occur, and therefore, Leuconostoc oenos dominates the wine. Pediococcus and Lactobacillus spp. will dominate as the pH approaches 4.0. Upon inoculation with a starter culture, a portion of the cell is killed immediately the extent of which depends on pH and temperature. The metabolic activity of LAB is also influenced by pH. Wine sugars are more readily metabolized at pH 3.6, causing an increase in volatile acidity. Diacetyl production is favored by low pH. Tartrate degradation is more likely to occur at pH above 3. 5.

Sulfur dioxide: Widely used as an antioxidant and to control wild yeast and bacteria. Total

S02 of 100-150 ppm or bound S02 of 50-100 ppm or free S02 of 1-10 ppm are sufficient to interfere with the growth of LAB in wines depending on the species, pH, and the amount of insoluble solids in the wine. It is important to distinguish between the amounts which retard growth and those which kill the cells. Lower pH values give a higher free molecular S02 and it is this form which has the greatest antimicrobial effect. Addition of20 ppm S02 at pH 4.0 decreased viability of Leuconostoc oenos by 15% after 2 hours, at pH 3.6 and 3.2, the viability decreased by 85% and 100%, respectively.

Alcohol: Survival and growth of LAB decrease as the ethanol concentration increases above 10%. Most spp. of Leuconostoc and Pediococcus appear to have maximum tolerance of 12% to 14% alcohol, and Lactobacillus of about 15% alcohol. But, Lactobacillus trichodes has been isolated from wines with 20% alcohol. Alcohol tolerance is affected by temperature and pH, and decreases as temperature and pH increase. It has been shown that malic acid degradation was not effected by 11% alcohol, but at concentration of 12% and 13%, the degradation was reduced to 56% and 16%, respectively.

Temperature: In wine the optimal temperature for growth of LAB and MLF is 18-20°C. MLF is strongly inhibited by low temperatures. Only a few Leuconostoc oenos species can conduct MLF below 15°C. MLF is rarely observed below 10°C. Also, temperatures above 25°C severely inhibit growth.

104 Vinification: Early and excessive clarification can significantly reduce the LAB population and thus, delay the onset ofMLF. Skin contact during fennentation and lees contact after fennentation promote nutrient release and thus, onset ofMLF.

Yeast-bacteria interaction: Some strains of Saccharomyces may inhibit LAB by production of metabolites such as S02 and toxic fatty acids, or by nutrient depletion such as amino acids. Yeast autolysis stimulates LAB due to the release of nutrients. Small amounts of carbon dioxide are also stimulatory to the growth of LAB. Leaving the wine on the yeast lees to maintain a higher level of carbon dioxide has been suggested.

Undesirable Growth: Where the growth ofLAB is not desired, the above mentioned factors affecting survival and growth should be taken into consideration. Generally, free sulfur dioxide of0.8 ppm as molecular inhibits growth. Processes contributing to minimizing the cell count such as settling/clarification, early racking, early fining/centrifugation/filtration are all helpful in minimizing the potential for later growth. Filtration through 0.2 to .45 t-tm membranes or pasteurization effectively prevent any growth.

Desirable Growth (MLF): Where MLF is to be used as a winemak:ing tool, close attention to conditions conducive to growth must be paid. The following parameters should be considered: suitability of grape variety, style of wine, method ofvinification, spontaneous or induced (strain) MLF. When MLF is desired, as many factors as possible should be optimized. The factors are interactive and should not be looked at in isolation. The induced MLF is by far today's preferred choice because ofbetter predictability and control. The key factors for a successful MLF are:

SQ2 level: Ideal= 0 ppm; maximum= 30-35 ppm as total; free less than 10 ppm. pH: Lower limit= 2.9-3.0; best= >3.1; upper limit= 3.4-3.5. Juice acid adjustments can be made with MLF in mind. Too high a pH will cause excessive diacetyl production. Too low a pH inhibits MLF.

Temperature: Ideal= 22-25°C; lower limit= l5°C. Inoculation should not be done at temperatures below l8°C.

Starter Culture: Several good technical papers are available on this subject. Always use a culture which is active and has a high cell count. Such a culture is characterized by a large number of cells in long chains.

Time oflnoculation: Ideal = when active primary fennentation begins or immediately at its completion.

A rapid MLF produces a more subtle character and has less impact on fruit flavors. The wine is more integrated and better balanced. Slow and extended MLF produce a less fruity style with strong butterscotch character.

105 BACll..LUS:

There are only a few literature reports about the presence of these organisms in wine. They have been isolated from wines with a relatively high alcohol content. One particular Australian study isolated these organisms from a bottle of contaminated Brandy with an alcohol content of 38%.

The sensory effect of bacillus contamination has not been established. However, it would appear that visual effects are more serious than the sensory alteration of the product. The source of the bacillus is more than likely from tap water and contamination associated with improper filtration and sanitation methods.

SUMMARY

Winemakers are generally doing a lot of things right when it comes to controlling bacterial growth, whether this being on the grape, in the juice, or during fermentation and subsequent storage of the wine. However, if our grape harvest, crush, fermentations and storage are done with little thought to bacterial activity, the wine will become affected negatively.

106 IMPACT OF MICROBES ON GRAPE PRODUCT QUALITY & MICROBIAL EVALUATION OF WINERY SANITATION PRACTICES

Ellen Harkness, Analytical Technologist Department of Food Science Purdue University, W. Lafayette, IN 47907-1160

The production of wine is still a magical and moderately unpredictable endeavor. Since grape must is not sterilized to kill all microorganisms, nor is a production facility ever sterile, no matter how thorough the sanitation process, every fermentation is a mixed bag - every wine a zoo of yeasts and bacteria fighting for available nutrients, each beast adding his own particular character to the final brew. The gist of this lurid description it to reassure you that every winemaker has made wines which smell of elephant droppings; but, the winemaker dedicated to quality control, with a good basic understanding of the behavior of the organisms involved, should rarely experience such a disaster.

Microbes are living organisms which can only be seen with the aid of a microscope. The ones which play a part in the final character of wine can be grouped into two separate kingdoms of the Five Kingdom Classification System, the Fungi (yeasts and molds) and the Procaryotes (bacteria). Bacteria are very primitive in that they usually require less complex nutrients, they have a simple reproductive system. Yeasts and molds are structurally more complex, and generally they are more tolerant of inhospitable environments. Fungi are active at low pH, they tolerate high sugar or salt concentrations, lower water content, less nitrogen and lower temperatures. These are generalizations; however, they are fairly accurate when limiting our microbial discussion to those associated with wine.

THE FUNGI

Filamentous fungi or molds will not grow in bottled or properly stored bulk wine, but they are often associated with vineyard bunch rot and sour rot. They will happily grow in any damp place with a bit of grape juice or wine left behind, such as hoses, barrels, air locks, and musty/moldy aromas have never been appreciated, whether in tennis shoes or wine. The main offenders have names like Penicillium, Aspergillus, Mucor, Rhizopus, Botrytis, and they are all detrimental to wine quality. Increased levels of glycerol, acetic acid, ethanol and gluconic acid are noticeable in musts extracted from rotten fruit. Botrytis is the fungus responsible for the condition we call . Infection occurring late in the season, followed by DRY conditions, may produce concentrated berry components by a wicking action. Since the mold produces gluconic acid, glycerol, galacturonic acid, attacks malic and even tartaric acid, the resulting wine will have very different flavor/aroma characteristics. Often the line between "rotten" rot and "noble" rot is very fine indeed. Under the best conditions, it is the only laudable mold associated with wine.

Yeasts are classified as Fungi although they have many noticeable differences. Microscopically they are typically single celled, oval or spherical, multiply by producing a

107 structure called a bud at some point on the mother cell which grows and finally splits off. Although some yeasts may produce filamentous looking structures, they are not the true hyphae with cross membranes produced by filamentous fungi. As many as 19 - 20 different yeasts have been reported occurring on the skins of grapes, the vast majority of which are considered undesirable in winemaking. There is a significant difference in the alcohol tolerance of different yeast species which accounts for the 11 sequential 11 nature of yeast populations in a fermenting wine. Various reports suggest that yeast populations at the beginning of all fermentations are mixed, comprised mainly ofKloeckera, and Saccharomyces cerevisiae, among others, narrowing to Saccharomyces as alcohol levels increased, but becoming more complex with the appearance ofHansenula, Pichia, Brettanomyces and other Saccharomyces species as fermentation finishes.

For winemaking and quality control purposes, yeasts can be divided into two groups based on their chemical behavior.

Fermentative yeasts are responsible for the conversion of sugar solutions to alcoholic solutions, a process which does not require oxygen. The primary fermentative yeasts are:

Kloeckera, a relatively small (2-4 11m) lemon shaped yeast, often the primary wild yeast in early fermentation. It may be associated with higher levels of acetic acid and aldehydes if its dominance is not challenged by the more desirable fermenter,

Saccharomyces cerevisiae, round, medium to large cells (7 x 8 11m), showing budding at ends. They are alcohol tolerant to 12-14% or higher, moderately tolerant to sulfur dioxide and have a wide temperature range tolerance. S. cerevisiae strains rapidly produce alcohol from sugar without adding objectionable odor or flavor components and are the yeasts selected for nearly all of the commercial starter cultures.

Brettanomyces is considered an undesirable fermenter since it is capable of producing large amounts of acetic, isobutryic and isovaleric acids, yielding a pungent mousy or horsey, sometimes metallic character to the wine. In general this yeast is considered a serious spoilage organism and most wineries are careful to avoid any growth in their wines. However, since Brettanomyces has been credited with some of the unique characteristics ofFrench Burgundy wines, some American winemakers are experimenting with carefully controlled populations in their wines. It is easily identified in culture by its relative resistance to actidione (cycloheximide), tolerating concentrations in excess of 100 mglliter. Some strains are fairly resistant to sulfur dioxide at standard use levels. Because of these tolerances this yeast can survive and slowly grow in bottled dry wines, causing increasing damage to the wine and limiting its shelf life.

Oxidative yeasts are those which depend on oxygen and are therefore undesirable in winemaking, unless you are a sherry producer. Low alcohol wines are more at risk, with the oxidative yeasts utilizing alcohol and sometimes organic acids as carbon sources, producing volatile esters and aldehydes. These compounds in very small amounts may add complexity to the bouquet of a wine, in larger amounts they will damage the freshness and fruit aromas. All of

108 these are capable of producing a chalky wrinkled white surface film if undisturbed for a long enough period in containers having some head space. They reproduce by budding but are also capable of producing filament-like structures in surface films. The most common oxidative yeasts isolated from wine are:

Hansenula, a medium to oval or oblong shaped yeast, is fermentative as well as oxidative since it can produce alcohol from sugar. It has been studied for its potential to develop desirable bouquet characteristics in inoculated mixed cultures.

Pichia, is usually a short rod like yeast, often very tolerant to sulfur dioxide and sometimes benzoic acid.

Candida, a distant relative of the infectious species Candida albicans, often produces long rod shaped cells, budding usually from the ends.

THE BACTERIA

Of the thousands of bacteria now classified, only 6 different genera have been found growing in wine. This is due to the nearly antiseptic combination of alcohol and . Not only do most organisms not grow in wine, they actually die off if introduced through contaminated water, human, animal, or soil contact. Microbiologically speaking, unknown wine is much safer than unknown water. Wine bacteria can be grouped into two camps: those which produce acetic acid (acetic acid bacteria) and those which produce lactic acid (lactic acid bacteria).

Acetic Acid Bacteria are small gram negative organisms. They can utilize alcohol (ethanol) in the presence of oxygen to produce enormous quantities of acetic acid (vinegar), called volatile acidity in wine since the acid can be steam distilled. Even minute quantities of oxygen will support the production of undesirable levels of acetic acid, which may be chemically oxidized to ethyl acetate. They are capable of utilizing various sugars remaining in wines to produce gluconic and ketogluconic acid in small amounts, they can metabolize glycerol to dihydroxyacetone, and they may metabolize (break down) citric, malic, succinic, and fumaric acid. Since organic acids may go on to form aromatic esters with various alcohols, the potential for aroma and flavor changes in a wine under acetic acid bacterial attack is extremely important and nearly unpredictable. Acetic acid is a component of all wines; however, the level at which it is damaging to a wine's quality varies with the wine and with the consumer. Although yeasts and some other bacteria do produce acetic acid, levels which have a noticeable sensory effect are usually the result of acetic acid bacterial spoilage. BATF regulations set the maximum allowable limit for acetic acid at 0.12gm/100ml in white wines, and 0.14gm/100ml in red wines. Acetic acid bacteria are tolerant of pH levels as low as 3. 0 but are more active at higher pH levels.

109 Two genera of acetic acid bacteria are found in wine.

Gluconobacter are small gram negative bacteria which may be nearly round or seen as long irregular strands. It differs from the other acetic acid bacteria in that it can only oxidize ethanol to acetic acid. Gluconobacter is the predominant acetic acid bacteria on grapes where levels of alcohol are very low. It is responsible for producing undesirable levels of gluconic acid from glucose in badly infected musts, before alcohol concentration reaches 3.5%. Gluconobacter can produce extracellular fibrils of cellulose from glucose, and may develop a thick tough pellicle on the surface of a must of low alcohol content. Even early stages of cellulose production could affect the filterability of a wine.

Acetobacter species appear identical to Gluconobacter microscopically; however, their tolerance to ethanol, in some cases up to 15%, assures that they are the dominant acetic acid bacteria in finished wines. They are separated from Gluconobacter by their ability to oxidize ethanol to acetic acid and then, under very low ethanol concentrations, on to carbon dioxide and water. Several strains of Acetobacter can also produce cellulose fibers.

Lactic Acid Bacteria are responsible for the process called malolactic fermentation by metabolizing malic acid, producing lactic acid and carbon dioxide. They do not require oxygen. The lactic acid bacteria are gram positive, extremely small, and very slow growing, compared to yeasts. They are also capable ofutilizing citric acid and glucose producing important flavor compounds including acetic acid, diacetyl and acetoin. At best, their activity can increase the complexity of a wine, reduce the acidity, and reduce the varietal fruit character. At worst, a flat, mousy, sauerkraut character can develop. Three genera oflactic acid bacteria can be isolated from grapes and wine.

Leuconostoc is the most common lactic acid bacteria isolated from low pH wines. Some will tolerate pH levels of 3. 0 and have been reported to induce malolactic fermentation in cuvee material with a pH of2.8-2.9. Microscopically it appears as pairs of cocci (spherical cells) in chains. Since it is a heterofermentative organism ( produces more than one end product), it also produces small amounts of acetic acid. Leuconostoc oenos is the main organism used for commercial malolactic starter cultures primarily because of its tolerance to low pH.

Lactobacillus are more often isolated from wines with higher pH levels. Microscopically they may appear as short to medium length rods, usually a bit larger than Leuconostoc. Since Lactobacillus grow more rapidly than Leuconostoc, some starter culture work has been done with this genus.

Pediococcus appears as clusters of cocci microscopically, and is easy to recognize. It is homofermentative, producing only lactic acid and carbon dioxide from malic acid, but is not used for starter cultures because of its sensitivity to low pH and high alcohol wines. In general, both Pediococcus and Lactobacillus are associated with off flavors and aromas - problems which may be more pH related than due to specific compounds produced by these bacteria.

110 FACTORS AFFECTING MICROBIAL GROWTH

As with all living things, the ability of microbes to thrive is directly dependent on their environment . • Moisture is absolutely essential to cellular activity. In extremely dry environments, actively growing cells will eventually die or go into a dormant state if they have that capability. As the .. moisture content of the environment increases, cellular activity increases. In situations where there is a very high concentration of dissolved solids (sugars or salts) living cells cannot utilize the moisture in solutions and are not able to multiply, thus jellies, concentrates and brine solutions are resistant to microbial spoilage.

Temperature is a controlling factor for microbial activity. Optimum growth rates for wine related organisms is in the range of 60-85 F; however, some yeasts are quite capable of fermenting slowly at temperatures as low as 28 F. Most microbial growth ceases at temperatures above 100 F, and viable cells (actively growing cells) die at temperatures above 155 -160 F, especially ifthey are exposed to that temperature for 3-10 minutes.

Nutrients are required by all cells, but each organism has specific and differing requirements. Wine provides carbohydrates in the form of sugars, ethanol and organic acids. Amino acids and ammonia are available in wine and juice as sources of nitrogen, and juices contain vitamins and other complex factors required for bacterial growth.

pH, a measure of acid strength, is a major factor controlling microbiological activity. Low pH is one of the main reasons so few microbes will grow in wine. Yeasts and a few Leuconostoc sp. will tolerate a pH as low as 2.8. Wine organisms are increasingly active as pH increases to 4.0. Since wines experience serious non-microbial problems at pH higher than 4.0, high pH is never a factor in inhibiting microbial growth in wines or juices.

MICROBIAL SPOILAGE OF FRESH JUICE

The primary problem plaguing fresh juice processors, whether packaged juice is the final product or whether attempting to hold fresh juice for later fermentation or wine additions, is yeast fermentation. According to FDA regulations, an excess of0.5% ethanol is illegal in a product labeled as juice. A secondary problem is Acetobacter spoilage producing increased levels of acetic acid.

Detection & Identification

The presence of gas in juice, as sugar is fermented to alcohol and carbon dioxide, is always evidence of yeast spoilage. As the spoilage progresses, sour smells and tastes develop. Bulk juice should be monitored for ethanol (dichromate method is needed). Juice samples should be cultured to determine numbers of viable yeast cells, or observed microscopically to identify budding yeast cells which indicates active growth.

111 Prevention

Sanitizing Equipment: Careful cleaning and sanitization of all surfaces coming in contact with fresh juice is absolutely necessary, whether tubing or tanks. Ifjuice is being preserved by sterile filtration, all bottles and closures must be sterilized.

To sanitize surfaces with heat, temperatures in excess of 161 F for 15 minutes are required. .. If possible, attempt to reach temperatures nearer to 2000f. Critical areas are tubing connections and rubber seals or gaskets, which may have crevices which are harder to clean and take longer to reach critical temperatures.

Chemical sanitization is effective for surfaces, but is less effective than heat for the flexible connections described above.

-Sodium hypochlorite (bleach) is an extremely toxic compound for microbes and will kill all fungi and bacteria (as well as viruses) on contact if the proper concentration of active material is available. Hypochlorite solutions are not very effective when soap or excessive dirt residue is present. It is destructive to wood surfaces, and makes obnoxious fumes, and must never come in contact with caustic soda (chemically basic solutions) or chlorine gas may be generated. For production floors, walls and work surfaces, as well as tubing or other juice contact areas, 114 cup sodium hypochlorite solution per gallon of water is recommended. Surfaces should be dried or rinsed with water before allowing juice contact.

- Sulfur dioxide in acidified water is a more juice compatible sanitizer and can be used safely for sanitizing barrels, corks, filter pads, as well as plastic and steel surfaces (although this concentration may pit stainless steel tanks with prolonged exposure). 250 ppm sulfur dioxide in a pH 3.0 solution will effectively kill most of the viable microbes in 30 minutes. It is not as effective as hypochlorite, but it is safer in case of product contact. Dissolving 1.3 grams metabisulfite and 12 grams of citric acid in water will give the 250 ppm concentration required, and heating the water makes it more effective.

Sanitizing Product: Sterile filtration as a method for preservation ofjuice and wine can be effective IF all filters and equipment have been properly sanitized, and IF the environment is free of contaminating microbes. Even with state of the art equipment, these criteria are hard to meet, and sterile filtration should be backed up with some chemical or heat treatment to guarantee adequate shelf life, especially with juice. For juice and wine sterilization, only a membrane filter of .45 micron or smaller pore size is effective. Depth filters can remove nearly all microbes, but not all, and are therefore not truly "sterile" filters.

Pasteurization ofjuice is a common and safe method for killing all microbes which could grow and spoil the product. For juice, pH less than 4.0, rapid heating to 190-200 F, filling heated containers, sealing, inverting gently for 3 minutes, then cooling as rapidly as possible, will effectively preserve bottled juice in an inexpensive manner with minimum heat damage to

112 the product. This is called "hot fill" and is good for the small producer since the heated product sanitizes the container and eliminates problems from air contamination in the production area.

Chemical preservation ofjuice is another effective method which is realistic and moderately safe for the small producer. Depending on the pH of the product, 50 ppm of free sulfur dioxide combined with 300-500 ppm benzoic acid (or combination of sorbic and benzoic acid which is thought to give a better flavor) will prevent spoilage ofjuice which has been filtered to reduce the yeast population to less than 10 cells per milliliter ofjuice. Iflarge numbers ofyeast are present in the juice, and the pH is higher than 3.2-3.4, the treated product may not be stable. This method is especially effective for carbonated beverages.

MICROBIAL SPOILAGE OF JUICE CONCENTRATES

When juice has been concentrated to at least 68'13rix, one of the basic necessities for microbial growth is eliminated: water. The concentrate itself is very resistant to spoilage under these conditions. One problem which can develop when concentrate is moved in and out of refrigeration is condensation of moisture on the inner surfaces of the container. This condensate will have some of the sugar and other nutrients from the concentrate in solution and may support yeast or mold growth on those moist surfaces.

Detection & Identification

When the concentrate is less than 68'13rix, slow yeast fermentation can occur with production of gas and reduction ofBrix. Some molds may also be able to grow on the surface of the concentrate, visible as fuzzy patches and producing musty smells when the container is opened.

Prevention

Juice concentrated to 60-70'13rix should be stored at 320f, and those concentrates at 40-65°Brix stored at 23°F especially when long term storage is anticipated.

MICROBIAL SPOILAGE OF BULK WINE

Although wine fermented to dryness is relatively resistant to microbial growth, it is still susceptible to problems from undesired malolactic fermentation, acetic acid spoilage, surface yeast and Brettanomyces growth. In all cases, the higher the wine's pH, the more likely microbial growth will occur, the faster it will occur, and the more damage will result.

Detection & Identification If

-Malolactic fermentation is accompanied by small amounts of C02, decreasing Total Titratable Acidity and increasing pH, and small but sometimes significant increase in Volatile Acidity. Paper chromatography indicates increasing lactic acid, and when the malolactic fermentation is complete, total absence of malic acid. Microscopic exam of the wine reveals large numbers of

113 bacteria throughout the tank, settling to the bottom when malic acid is depleted. Growing lactic acid bacteria is not difficult if enriched media is used. Modified Rogosa Agar is the most common, with 100 ppm of cycloheximide added to inhibit yeast. Most lactic acid bacteria take 4-8 days to produce visible colonies.

-Acetic acid spoilage (vinegar) results in wine with sour taste and smell, slimy to rubbery surface film when spoilage is advanced, increased Total Titratable Acidity, and very large increases in Volatile Acidity. Microscopic exam of the surface film reveals gram negative bacteria. Culture of these organisms is sometimes difficult and usually unnecessary since the large increase in volatile acidity is diagnostic.

-Surface yeast growth is observed as a powdery, sometimes blistered film on the surface of wine and is easily diagnosed using a microscope. In early stages surface yeast growth may be confused with acetobacter surface film; however, it is easily identified microscopically.

-Brettanomyces growth is slow and subtle and often goes undetected until the wine is damaged by unpleasant odors and flavors. It does not necessarily produce gas or a surface film, can damage wine even at fairly low concentrations although recent research suggests that wine may not be ruined until the population of Brettanomyces reaches one million cells per milliliter. Microscopically it produces some lemon shaped cells but in general resembles Saccharomyces and requires culture techniques for final identification. The ability ofBrettanomyces to produce creamy white colonies after 4-7 days on Yeast Mold Agar with 100 ppm cycloheximide is diagnostic.

Prevention

-The single most important factor in maintaining quality in bulk wine is the ability to maintain storage containers absolutely full. Flushing head spaces with inert gases such as C02 or N2 helps for short term storage but is risky for long storage without sophisticated gas metering equipment.

-The colder the storage temperature, the longer it will take for microbial damage to occur.

-Maintain free S02levels appropriate for the pH of the particular wine.

-When dealing with surface yeast and acetobacter spoilage, filter the wine through at least .5 Jlm nominal filters into sanitized containers, adjust S02 and bottle as soon as possible. Surface yeast spoilage is particularly difficult to discourage and often reappears, even after filtration, due to winery contamination. .. MICROBIAL SPOILAGE OF BOTILED WINE

In a properly filled and sealed bottle, acetic acid bacteria are rapidly incapacitated and do no further damage to the wine, even if chemical preservatives are inadequate. The most common

114 microbial problem in bottled wine is yeast refermentation ofwines with some residual sugar. Haziness with large amounts of gas causing gushing of the wine when the cork is pulled is characteristic of yeast spoilage. Some yeast can grow slowly in the bottle producing sticky clumps which settle rapidly when disturbed. Brettanomyces are capable of growing slowly after bottling, rarely producing more than a small fine sediment even after several years, but causing deterioration of aroma and flavor. Malolactic bacteria can grow rapidly in corked bottles producing hazy wine and small amounts of gas, usually seen as slowly rising fine bubbles coming steadily to the surface after the cork is pulled.

Identification

In all cases, microscopic exam of the bottle sediment will reveal the culprit. Brettanomyces is positively identified by its ability to grow on agar containing cycloheximide.

Prevention

-Free S02levels slightly above the amount required to provide 0.8 ppm molecular S02 at the wine's pH will prevent bacterial growth in bottled wines.

-When residual sugar levels are higher than 0.2-0.3%, use 180-200 ppm potassium sorbate (usually I gram per gallon wine) along with appropriate levels of sulfur dioxide to prevent yeast growth.

-Heat may be used to stabilize wine in the same manner as described for juice. It is rarely used since delicate wine flavors are easily damaged during heating.

-Sterile filtration without any use of preservatives at final bottling is risky but can be absolutely effective if the filter and all tubing, containers and closures as well as the bottling environment are properly sanitized. When using this method, it is especially important to evaluate bottled wines for sterility in order to head off pending disaster.

IMPACT OF THE GOOD GUYS

All commercial yeast starter cultures must be capable of the following:

- ferment to dryness at 32-100 F - be active in the presence of 50-75 ppm free S02 in must - produce low levels of S02 and H2S -tolerate 30-38% sugar -tolerate pH as low as 2.8 - tolerate 14% alcohol

115 Special commercial yeast starters are capable of:

- producing killer factor to eliminate other wild yeasts - reduce malic acid to help high acid wines - increase monoterpene flavors in certain wines - clump and settle out for sparkling wine production - produce up to 18% alcohol under certain conditions.

Commercial malolactic cultures can:

- tolerate pH 3.2 - convert malic to lactic acid - lower total acidity by about 25% -add complexity to flavor, diacetyl and acetoin, especially when malolactic fermentation takes place after yeast fermentation is completed -reduce varietal character, fruitiness - improve bottle stability - not produce unpleasant off aromas of sauerkraut or mousiness

NO-BRAINER TIPS FOR YEAST AND MALOLACTIC STARTER CULTURES

- buy fresh each season from reliable major supplier - store all cultures refrigerated all the time, malolactic cultures keep longer when frozen - buy enough to inoculate all anticipated must, too much is better than running out when special reserve premium winemaker's select harvest is ready to inoculate - choose one yeast strain for warm red fermentations, one for cool white fermentations. Unless you are extremely careful yeasts "visit" other tanks than the one they were introduced to, consequently efforts to evaluate many different strains may not mean much - if you use any killer strains, use all killer strains -FOLLOW SUPPLIER INSTRUCTIONS FAITHFULLY FOR BEST RESULTS - resist using finished fermentations to inoculate new must -use yeast nutrient (Fermaid, Yeastex) at 2#/1000 gallon rate to help avoid stuck fermentations - be sure must temperature is within 5-10 degrees of starter culture or rehydration at time of inoculation

MICROBIAL EVALUATION OF WINERY SANITATION PRACTICES

Whether or not you like to admit it, if you are dependent on controlling the behavior of yeasts to make your living, you are a microbiologist. Fortunately most of the microbes encountered in a winery are involved in performing desired services such as making alcohol out of sugar, and turning malic acid into lactic acid. The encouragement and manipulation of these elite microbes is detailed in the literature distributed with commercial cultures and is generally

116 well known to winemakers. However, enologists are also constantly harassed by microscopic villains, and this presentation is designed to give you some insight in dealing with their detection, identification and control. Although lack of equipment and familiarity with techniques may limit your range, many important microbial matters can be properly attended to by the layman.

You will need to begin with a clean white lab coat and a few bits of micro trivia to impress your visitors. Try these:

1. Wine yeasts replicate mainly by a process called budding, which, under optimum conditions, may result in doubling the population every two hours. Therefore, a single lonely yeast may create a world of more than 16 million relatives in one wild weekend.

2. Wine may appear visibly clear while hosting a party of 100,000 bacteria or 10,000 yeasts in every milliliter. That is 50,000 yeast cells in a teaspoon of wine.

3. One gram of dry yeast contains 10 billion living yeast cells. Think about that next time the can tips over in your refrigerator.

4. No infectious microorganism, whether yeast, bacteria or even virus, can grow or even survive very long in the hostile environment of wine.

A WORKPLACE

The ideal place for your microbiological activities would be a small room, bathroom size, with washable walls, ceiling and floor, and containing a small sink and formica table top. Put a sign on the door limiting use and access to certified microbiologists (wearing white coats) only.

Second choice would be a corner of a multipurpose room, away from the fermentation area, which could be isolated from visitors, workers and outside air currents when micro work is in progress.

A third, and very weak choice, would be a corner of the winery.

When microbiological tasks must be performed in a multipurpose area, a small contained workplace called a hood, which can be sanitized and will protect the work area from dust and air currents, is necessary. If you purchase used equipment avoid chemical hoods, as they are designed for rapid air circulation in the wrong direction for micro work and are impossible to sanitize. Plans for building a small effective hood are illustrated in Figure 1. This unit, made of 1/2 inch plywood, 1/4 inch plexiglass and a few hinges, costs less than $50 to build. The plywood back and adjoining sides are cut on a bevel and hinged together to collapse for storage. The plexiglass panels which give the user visibility, while deflecting dust and breath aerosols, are slid into grooves made by 1/4 inch chair railing strips. The unit is covered with several coats of white, high gloss, appliance enamel paint.

117 A small styrofoam incubator (Fig. 2) will provide a continuous temperature to grow yeasts and bacteria rapidly. The temperature is controlled by the wattage of the light bulb used in a socket assembly which has been inserted through the side of the styrofoam ice chest. This unit maintains a constant temperature of75° F with a 5 watt bulb and costs less than $10 to assemble.

The following list contains most of the equipment needed to collect if you plan to set up a small effective micro laboratory. A source, catalog number and approximate price for all items are listed; however, in most cases they are available from many different suppliers who will be glad to cross reference catalog numbers. Sources listed here are based mainly on price and availability.

EQUIPMENT:

1. Hood - specifications listed below

2. Incubator- specifications listed below

3. Aspirator Filter Pump- Nalgene. Fisher Scientific, Cat. # 09-960-2, $5. Produces vacuum needed to pull wine samples through filter assembly.

4. Alcohol Burner- glass lamp. Fisher Cat# 04-245 AA, $4. Used to bum off alcohol on disinfected equipment, dry microscope slides, sterilize loop, etc.

5. Side Arm Vacuum Flask- 1000 ml Nalgene. Fisher Cat.# 10-fl82-50B, $11. Supports filtration unit for sterility evaluation ofwine andjuice.

6. Rubber Stopper, size 8, one hole. Fisher Cat.# 14-135M, pkg 12/$12. Connects filtration unit to vacuum flask.

7. Vacuum Tubing, black rubber. Fisher Cat.# 14-1750, pkg. 12 ft/$35. Connects vacuum flask to sink aspirator.

8. Millipore Yeast & Mold Swab Test Kits. Millipore Cat. # MYSK 0000 25, 25 tests/$100. Used to evaluate microbial contamination ofwinery equipment, corks, etc.

9. Millipore 55 Plus Monitor. Millipore Cat. #MHBG 05500, pkg 50/$81. For analysis ojyeast, mold and bacterial contamination ofjuice and wine.

118 10. Millipore Media in 2 ml plastic ampules: Added to 55 PLUSfilter monitor after sample filtration to provide nutrient for microbial growth. Tomato Juice Broth, Cat. # :MXOOTJ220, pkg 20 ampules/$22 Best for lactic acid bacteria, also yeast and mold WL Nutrient Broth, Cat. # MOOOOOP2N, pkg 50 ampules/$42 Yeast, mold and bacteria. WL Differential Broth, Cat. # MOOOOOP2D, pkg 50 ampules/$45 Contains cycloheximide to inhibit yeast growth, except Brettanomyces.

11. Paper Chromatography Kit. Presque Isle Wine Cellars Cat. # PCK-2V, #31. Detects malolactic fermentation in wine.

12. Methylene Blue. Fisher Cat.# M281-25, 25 gms/ $25. Dissolve one gram Methylene blue powder in 10 mls alcohol, add 90 mls water. Use to stain yeast and bacteria for microscopic observation.

13. Alcohol, denatured ethanol or isopropanol. Drug store item. 70% solution in water good disinfectant for surfaces that will come in contact with wine. Denatured is fine for culture work, use food grade for corker jaws, filler spouts.

14. Sterile Whirl Pack Bags- 180 mi. Fisher Cat. # 01-812-5A, pkg 500/$36. Sterile, disposable plastic bags for sample collection from tanks, barrels, etc.

The purchase of a microscope is not within every winery's budget; however, if you do not have access to a university or medical laboratory with a good microscope to help you evaluate wine sediment problems and identify microbes, there are several alternatives. If you are fortunate enough to find a reasonably priced used microscope in good condition with built in illuminator and oil immersion capabilities, buy it. Many universities sell surplus microscopes which have become obsolete, but are more than satisfactory for these purposes. Although phase contrast microscopy is often discussed and can be helpful, the microscope is vety expensive and requires a thorough knowledge of microbes to differentiate them from debris in the sample.

15. New & Reconditioned Microscopes. Spectro Services, Inc. One reliable source of used microscopes with warranties.

16. Microscope accessory pack. The Wine Lab, Cat. #1M880, $25. • (Practical assortment ofglass slides, cover slips, lens paper, inoculating loop, etc.)

SUPPLIERS: MILLIPORE, PO BOX 255, BEDFORD, MA 01730 (800)645-5476 VINQUIRY, 16003 HEALDSBURG AVE., HEALDSBURG, CA 95448 (707)433-8869

119 THE WINE LAB, 477 WALNUT ST. NAPA, CA 94559 (707)224-7903 PRESQUE ISLE WINE CELLARS,9440 BUFFALO RD, NORTH EAST, PA 16428 (814)725-1314 FISHER SCIENTIFIC, 1600 W. GLENLAKE AVE., ITASCA, IL 60143 (800)766-7000 SPECTRO SERVICES, INC., 1653 EAST MAIN STREET, ROCHESTER, ~ 14609 (716)654-9500

MICROBIOLOGICAL PROCEDURES

Utilizing the equipment listed above and a few basic techniques, you should be able to analyze the major contamination problems in your winery.

Winery Sanitation

Evaluating the effects of winery sanitation procedures should begin with a careful visual and olfactory examination of the area, keeping in mind that one who lives by the sniff test may die by the sniff test. If you see dirt, cork particles, old dried mold residue in tubing, grape skins in the drain, sticky juice on filter drip trays, or if you smell off odors in tubing, carboys, barrels, trash cans, the area is not sanitary, no matter how much spraying of disinfectant may have been done. If your area passes an eye and nose inspection, then it's time for a microbial culture.

The Millipore Company produces a 'Swab Test Kit' with 'Yeast & Mold Tester' which is effective in determining the population of living yeasts and molds on a surface.

The first step in any microbiological procedure is to eliminate yourself as a source of contamination as much as possible. Putting on a clean lab coat laundered in hot water and clorox to cover clothing which may be spattered with wine or dirt, controlling long loose hair in a clip, and washing hands with soap, then spraying with 70% alcohol would all be effective. If the directions supplied by Millipore are followed carefully, you will have an accurate estimation of contamination of areas such as tank interiors, valves, corks, filler tubes, etc. The methodology involves rubbing the area in question with the swab, transferring the swab to a buffer solution to suspend any organisms which have been picked up, then dipping a media impregnated filter membrane into the buffer. The filter membrane is shaken to remove excess buffer, then replaced in its sterile housing and incubated, filter surface down, for 5-7 days at 75° F.

Look for growth after 48 hours, since mold colonies may grow fuzzy and very large obscuring the entire filter after a few days making it impossible to evaluate other growth. Each viable organism which lands on the filter will begin to multiply and eventually produce a small drop-like pile called a colony. Counting the number of colonies gives an estimate of how many living organisms were picked up on the swab. The presence of yeast and/or mold contamination indicates bacterial contamination also exists in the area tested, and the absence of yeast or mold growth gives reasonable assurance that the area is also free of bacteria.

120 In general, yeasts are smooth, shiny or creamy spots, 2-4 mm in diameter, growing in 2-5 days. Molds will begin as very small rough colonies becoming larger and fuzzier with time. They may be green or olive or black if spores form. Most bacteria will not grow on this kit. Occasionally Bacillus spp. from dirt may grow forming yeast-like colonies, or appear as a slimy, flat, rapidly spreading white or nearly transparent film on the tester membrane. If the results of your swab culture show more than 2-3 colonies of yeast or mold, better sanitation methods would be suggested. If your tester shows no growth in 5-7 days, give your sanitary engineer a raise.

Bottle Sterility

Although there are many different units designed for measuring living or viable organisms in a product, the Millipore 55 PLUS monitor is easy to use and designed to reduce accidental contamination. This system allows you to filter a volume ofjuice or wine - trapping any yeasts or bacteria on the surface of a filter membrane. When nutrient media is applied to the membrane and the system allowed to incubate at 75° F for 7-10 days, the organisms will grow into colonies which can be enumerated and identified.

Careful washing of the bottle to be evaluated, and spraying the neck, cork and cork puller with 70% alcohol will eliminate most of the potential for contamination from the exterior of the bottle. The "Ah-So" type of cork puller with two prongs, used carefully, generally produces fewer cork crumbs than the screw type. Shake the bottle thoroughly just before removing the cork or and pour 100 ml of wine into the filter unit. If the wine is an unfiltered or very intense red, begin with 25 ml to be sure the entire sample passes through the membrane. Millipore furnishes extensive directions for the use of these units. Sterile water needed to rinse the excess wine (and preservatives such as S02 and sorbate) from the membrane may be obtained easily by microwaving a glass bottle with a microwave safe plastic cap half filled with water. Allow the contents of the bottle to come to a boil, then keep it simmering for 5 minutes on low power. As soon as you remove the bottle from the microwave, tighten the cap and allow the contents to cool.

Evaluation of the colonies growing on this filter system is similar to the 'Yeast and Mold Tester' in many ways; however, the WL nutrient medium does allow growth of wine spoilage bacteria and gives some color differentiation of the colonies.

In general, if a colony growing on a filter membrane is larger than 7-1 Omm after 7 days, slimy, fuzzy or exotically colored (red, pink, orange shades), it is not a wine spoilage organism. Since molds will not grow in bottled wines, they are not a concern in this evaluation. The presence of mold colonies or other non-spoilage organisms may indicate poor techniques during wine sampling, in which case several bottles should be selected for re-testing.

Yeasts will produce visible colonies in 3-5 days as described above. Bacteria take 5-l 0 days, colonies will appear very small, sometimes nearly transparent requiring magnification to identify them. The presence of one or less colonies on the filter after 10 days gives you a great

121 deal of confidence that your wine is microbiologically stable. Even in a very dry wine, <0.1% sugar, the growth of a few yeast or bacteria colonies suggests that you should hold the wine for a few weeks at ambient temperatures and do another filtration evaluation to see if the population is increasing. If you count more than 10 colonies on the filter, immediate action should be taken before bottle spoilage goes any farther.

One good system for evaluating a particular bottling would be to sample the first bottle off the line (to determine whether cleaning procedures were adequate), the last bottle (to determine the integrity of the system) and several at regular intervals during the entire run. The middle samples give some idea of the extent of a potential problem.

Commercial laboratories charge $10-20 for this type of analysis, depending on your requirements. The Millipore kits cost about $1.50 per sample and eliminate the need to mail samples.

When information gained from using these simple microbiological techniques is combined with organoleptic observations and chemical analyses, the causes of most wine spoilage can be determined in the small winery (fig. 3). If more information is needed, wine samples or cultures can be sent to commercial labs or universities with wine research and/or extension facilities for final identification.

Although there are many more techniques which can be acquired to do thorough microbial analyses of winery problems, I hope that these tips will help you feel more confident in doing some of your own evaluations and controlling the unwanted microbe activity in and around your wines.

122 INFLUENCE OF WINE COMPOSmON ON FILTRATION­ DIATOMACEOUS EARTH AND PAD FILTRATION

Kent R. Glaus Portage Hills Vineyards, Suffield, OH

Introduction

Wine filtration is one of the most important cellar activities that engages the winemaker. The reasons we utilize filtration technology include: the production of visually pleasing wines, the production of ":fiuity" wines, the production of physically and microbiologically stable wines, and for economic considerations in the recovery of wine from lees. Wine is a complex mixture of water, alcohol, particulate solids and colloids, and a host of up to 1300 other molecules and chemical complexes which give each wine its distinctive flavor, aroma, and stability. Wine filtration involves the chemical and/or physical interaction of some of these wine components with the filtration media. The choice of a particular filtration media therefore depends upon the objectives of the planned wine filtration and upon the particular composition of the wine to be filtered.

All wine filtration has one simple objective: removal of solids (such as yeast and bacteria) or macromolecules (such as colloids and polymers) before they can present an organoleptic or ascetic (visual) problem in a wine. There are two basic technologies employed within the wine industry to achieve this objective: Depth Filtration and Membrane Filtration. The importance of membrane filtration for the production of microbiologically stable wines (and the advances occurring with cross-flow membrane technologies which may soon obviate the need for depth filtration) cannot be overemphasized. A discussion of wine membrane filtration technology is contained elsewhere in these Proceedings so only the most important considerations in using and choosing depth filtration media will be discussed below.

Depth Filtration

There are two basic types of depth filtration universally employed within the wine industry -Diatomaceous Earth (DE) filtration and Pad filtration. Each of these two types of filtration have many different variations and methodologies that have been developed and marketed by a large number of commercial wine filtration manufacturers. However, these two types of filtration (and all of their commercial variations) have in common the following characteristics and properties:

1. Solids and macromolecules are retained within the matrix of the filtration media.

This characteristic actually defines the term "Depth Filtration". The matrix of each of these filters can have rather large volumes that can be defined in terms of area and thickness (or depth). It is within the large volume of these filtration matrices that the

123 solids and macromolecules in wines become entrapped.

2. The filtration matrix has high particle retention capacity.

This follows from the first basic characteristic of these filters - that is, the filtration matrix has a relatively large volume and consequently has a large capacity to hold the solids and macromolecules in the wine.

3. The filtration media can only be assigned a nominal retention rating.

Because of the particular physical arrangement of the filtration matrix in depth filters it is possible (and probable) that some solids or particles in wine can still pass through the matrix. Thus, the filter cannot "absolutely" filter out all solids or particles from wine. The efficiencies of depth filters are most often rated in terms of percent effectiveness. For example, a media rated for 98% at 1 micron means that we can be reasonably sure that 98 % of anything 1 micron or larger has been removed from the wine.

4. The efficiency of retention is dependent on flow rate and differential pressures.

The relative efficiency of a depth filter is largely dependent on the flow rates and differential pressures that are used during the wine filtration. These parameters vary somewhat and are in part determined by the specific type of filter, brand of matrix and manufacturer. Any deviations from manufacturer's suggested maximum flow rates and differential pressures can dramatically decrease the efficiency of filtration.

5. Separation of particles from wine occurs by mechanical sieving.

It has already been stated that depth filters have a high particle retention capacity. The solid particles in wine become entrapped within a complex and tortuous series of channels and passages within the filtration matrix. In DE filters this tortuous series of passages is self-forming during the filter coating and loading~ in pads, the passages have been formed during the manufacturing process. The entrapment of particles in these matrices is purely a physical event. Making the passages smaller (by choosing different DE grades or pads) allows smaller particles to be removed from the wine.

All Depth filters share these characteristics and properties but the similarity between DE and Pad filters ends here. While DE filters function by mechanical sieving alone, Pad filters have the ability to function by both mechanical sieving and by a chemical process called Electrochemical Adsorption. Due to the phenomena of electrochemical adsorption, pad filters have the ability to add a degree of physical stability to a wine that is not possible with a DE filter alone.

124 Electrochemical Adsorption and Wine Composition

Most commercially available pads have integral components which possess a net positive charge when exposed to wine (with a pH of3- 3.5). These components date to the period of the old asbestos containing pads - but in newer pads the asbestos has been replaced with binders and resins which acquire a similar positive charge. This is a very relevant and important consideration in wine filtration because most major wine particulates and colloids possess a negative charge in the wine environment. Major components of wine which possess a net negative charge generally include yeast and bacteria and two classes of macromolecules called polysaccharides and pectins. The notable macromolecular exceptions are many of the most troublesome (which can lead to hazing and which have a net positive charge in the wine environment). The difference in net charge between the pads and the negatively charged wine components results in electrostatic attraction between the opposing charges and the charged particles in the wine which are very efficiently adsorbed to the pads.

Many polysaccharides and pectins, both soluble and colloidal, can cause physical stability problems in a wine long after bottling. Furthermore, the presence of high concentrations of polysaccharides and pectins in a wine can often lead to premature blocking of subsequent membrane filtrations. Removal of many of these potentially troublesome macromolecules prior to bottling by a simple Pad filtration can avoid many of these unpleasant surprises before and after bottling. However, there are two considerations to keep in mind when contemplating a pad filtration for the removal of problematic macromolecules:

First, only negatively charged wine components are effectively adsorbed by the pads. As mentioned above, many of the winemaker's most problematic macromolecules are proteins and many, but not all, proteins have a positive charge in wine and are not electrostatically attracted to the filtration matrix. For these types of positively charged proteins, a routine fining of the wine with bentonite prior to either DE or pad filtration will agglomerate any positively charged proteins and allow their removal by mechanical sieving.

Second, the maintenance of a positive charge in and on the filtration matrix is dependent on a dynamic flow of wine through the pad filter. If the filtration is stopped for any reason mid-way through a filtration run then the electrostatic equilibrium that exists between the wine, the filtration matrix, and the wine macromolecules can be disrupted. This can result in a sloughing of the macromolecules back into the product stream following resumption of filtration. To avoid this problem during start-up (after an extended filter shut-down) the product stream can be briefly redirected or bypassed to the feed stream until the electrostatic equilibrium is reestablished . • Other Considerations in Depth Filtration

One principle that can not be overlooked in using depth filters is that larger particles are always removed much more efficiently than smaller particles. Since depth filters only have nominal retention ratings this is an important consideration. Anything the winemaker can do to

125 enhance the aggregation or agglomeration of smaller particles and macromolecules into larger particles and macromolecules will necessarily increase the overall efficiency of the depth filtration.

Fortunately, most macromolecules (both soluble and colloidal) are less soluble at lower temperatures than at higher temperatures. This means that the problematic macromolecules in wine will be more highly aggregated at a low wine temperature than at a higher wine temperature. The depth filtration of the wine at a very low temperature, as might be found immediately after cold stabilization, will greatly enhance the efficiency of the filtration.

Also, the purpose of most fining procedures is to agglomerate smaller particles into larger particles. Therefore, fining a wine with many of the more common fining agents will also drastically improve the efficiency of a subsequent depth filtration.

Problems in Depth Filtration

There are a few problems with depth filtration that the winemaker must keep in mind.

1. Surface blocking

Due to the elastic nature of the larger wine macromolecules, like grape debris, yeast, and bacteria, the surface of depth filters can be quickly blocked by the accumulation of larger particles on the active surface of the depth filter. This is less of a problem with self-forming DE matrices but can be a chronic problem with pad filters during course filtrations. When the surface of the filter matrix is blocked due to accumulated debris on its surface then the interior of the matrix is no longer available for mechanical sieving. This blocking , at least in the case of pads, is most often irreversible and can have significant economic consequences on a filtration regimen. For this reason DE depth filtration is most often the .filtration of choice for very course filtrations.

2. Media Migration

Due to the basic physical nature of both DE and pad filter matrices debris from the filtration matrix itself (DE particles, cellulose fibers, etc.) can often find their way into the filtered product stream. This sloughing or migration of the filter media is most often encountered during drastic changes in differential pressures due to frequent starting and stopping of a filter and can be minimized by a simple bypass or redirection of product stream into the feed stream following a filtration start-up.

3. Void Volumes

Almost all DE and pad filters suffer from a problem of rather large void volumes. The void volume is the volume that lies within the depth matrix itself For example, in a pad filter the initial void volume of the pad matrix is occupied by air; upon completion of a

126 filtration the void volume is occupied by residual wine. Depending upon the size ofthe filter the void volume can be quite significant and the loss of this wine retained within the filter matrix can be quite significant. The solution to recovering the void volume is to flush the depth filter with either water or an inert gas such as nitrogen . • 4. Microbial Growth

With proper care and under non-extreme conditions depth filtration media (both DE and pads) may be capable of supporting filtrations of several different wines extending over several days (or even weeks.) This type of usage lends itself to the possibility that contaminating microbial growth can develop within the matrix when the media is not in use, with resulting downstream contamination when the media is used again. The only way to avoid microbial growth over the extended life of the media would be to steam sterilize the media after use. If this is not practical or feasible then a hot water wash should be attempted for short term storage.

Choosing a Depth Filter

Depth filters are the most common filtration technologies in use in commercial wineries and depth filters are the primary means to achieve clarification and physical stability in a wine. The choice of a depth filtration technology for a wine should be guided by economic and labor considerations as well as by a basic knowledge of a wine's general particulate and macromolecular composition. However, because of their nominal retention rating, depth filters should never be counted upon to provide microbiological stability in a wine. To achieve microbiological stability it is imperative that a winery consider an absolute membrane filtration sometime prior to bottling.

In the ideal situation a winery would have both a DE filter and a pad filter available for depth filtration, and a membrane filter for absolute filtration. For rapid and economical course clarification of wines DE filters are often preferred over pad filters. However, while very tight grades of DE are now available (providing nominal sterile ratings) DE filters cannot provide the degree of physical and chemical stability in a wine (without pre-fining) that can be achieved by most common pad filters and pad media. Therefore, many wineries utilize a DE filter for course filtrations and pad filters for finer and pre-membrane filtrations. Smaller wineries that cannot afford the capitol costs of two different depth filters must carefully consider the relative costs and benefits of DE and pad filtration technologies~ for a smaller additional capital investment the best choice for a small winery is often a simple plate and frame pad filter with additional plates and dosing capability for DE filtration. Consultation with the host of domestic and foreign manufacturers of depth filters will reveal a vast number of different depth filter configurations and possibilities.

127 INFLUENCE OF WINE COMPOSmON ON FILTRATION­ ACHIEVING MICROBIOLOGICAL STABILITY THROUGH MEMBRANE FILTRATION

Peter Meier, Marketing Manager Millipore Corp, Bedford, MA

Membrane filtration is the premier method for achieving microbiological stability in the bottle. No chemicals and, of equal or greater importance, no heating is required. The organoleptic properties are preserved in a completely natural state until the product is opened, be it weeks, months or years after filling. Successful aseptic* filling requires a 'sanitary mind set'. Included are the location, sizing, design, operation, and monitoring of the entire system. The upstream fining and clarifying steps, the membranes which actually remove the microbes, the filling operation, the bottles and the closures are all important aspects.

The plumbing, the auxiliary fluids (such as counter pressure gases) to the filler, the containers, and all associated equipment require close adherence to standard sanitizing procedures and careful monitoring.

For over two decades, large and small bottlers of wine, coolers, juices, and beer have used membrane filtration as a part of their aseptic filling system. Several rules and guidelines for success have evolved over this period.

*1) aseptic is defined as a) preventing infection and also b) free or freed from (undesirable) microorganisms.

Membrane Filters

A membrane filter is a thin (0.1 mm) sheet of polymeric film with carefully controlled porosity (percent void volume) and pore dimensions. Polyvinylidene fluoride, nylon, and cellulose esters are among the plastic materials from which membranes are made.

Membrane filters retain microorganisms predominantly by a sieving action. Common pore size ratings are 0.22, 0.45, 0.65, 0.8 and 1.2 micrometers (hereafter called 'microns').

These ratings were established according to the membrane's microbial retention performance. Thus, 0.22 micron membranes quantitatively retain very small bacteria like Pseudomonas diminuta, and 0.45 micron membranes will quantitatively retain slightly larger bacteria such as Se"atia marcescens, Leuconostoc oenos, and Lactobacillus brevis. Quantitative retention usually implies at least 10 9 or 99.9999999% reduction in the concentration of microbes downstream of the membrane.

Membrane filters are commonly supplied as cartridge elements in lengths of 10 to 40

128 inches. Each 10-inch cartridge or equivalent (shown in J;'ig. 1) usually contains from five to eight square feet of surface area in a pleated configuration.

The flow is from the outside to the inside, and the filtered beverage exits the cartridge • through the open-ended throat which is secured into a housing with one or more o-rings or gaskets (Fig. 1) .

Fig. 1. 10-inch pleased final-membrane cartridges with common end-cap fittings.

Although the cartridge style is the most common configuration, flat sheets of membrane and newer sinall devices like stacked-disk units are also available (Fig. 2) .

.a;_;;e i ·ZM .Im.•~ Fig. 2 Convenient stacked-disk filters (also supplied in pre-sterilized and disposable configurations) provide a filter surface well-suited to small flows or small bottling runs.

129 Because some spoilage organisms are smaller than one micron--acetic acid and lactic acid bacteria, for example--most wine and beer bottlers will choose a final filter with a rated pore size of0.45 microns.

Larger pore sizes give higher through.;. puts, but they will not match the bacterial retention • properties of a 0.45 micron membrane. A more open pore size would be chosen only to insure retention of spoilage yeasts and molds (not all bacteria), or to achieve a high degree of clarity.

Although membrane filters have relatively low 'dirt-holding' capacity and are more expensive than pads or prefilters, their performance described above justifies the cost. Since the pore structure of a membrane guarantees absolute hold-back of undesired particles or microorganisms, it can be regarded as an 'insurance policy' for the user.

One way to keep costs down is to be sure that the beverage introduced into a membrane filter is reasonably free of plugging contaminants and microorganisms.

Even though a beverage may have sparkling clarity, it can plug a tight membrane quickly if it contains colloidal carbohydrates, for example, as in late-harvest wines. Pretreatment is accomplished by upstream fining operations, and by pad and/or diatomaceous earth filtration. Sometimes, a special set of cartridge filters (commonly known as 'prefilters') is used just before the final membrane.

Prefilters often have nominal or approximate pore size ratings and are placed immediately upstream of the final membrane to remove any remaining colloids. The nominal ratings for prefilter cartridges are usually much closer to absolute ratings than are the nominal ratings for diatomaceous earth and inexpensive depth filters, i.e., some pads or string-wound or molded cartridges.

Prefilters are carefully selected and tested by the supplier to improve the overall economy of the process. Suppliers of membrane filters will usually recommend methods to test the 'filterability' or the expected membrane through-put of the beverage*. If such tests show the presence of colloids which may prematurely plug final membranes, a prefilter or a very tight pad is the best choice.

*Such as that described by Y. Pe/eg and C. Brown, J. Food Science 41, p. 805 (1976). A 'filterability' test can often identify a beverage containing an excess ofplugging constituents. Additional upstream treatments, such as fining or tighter prefi/tration, may greatly improve filter throughputs and hence overall economy.

SIZING

Because membrane filters offer a fairly high resistance to flow, differential pressure is an important sizing consideration for both final membrane and prefilter cartridges.

130 Also, the rate at which membranes plug is always dependent upon 'face velocity'. This is the rate of flow per unit area of filtration media. The greater the face velocity, the faster the plugging.

To understand the effect of face velocity, visualize the pouring of rice through a coarse sieve. At a rapid flow, the kernels may bridge the openings and prevent passage. At a slower 'face velocity', the kernels will not bridge, but will flow cleanly through the openings.

(This is not to infer that slower flow rates allow microorganisms to pass through. The pore size of a final membrane filter prevents larger particles, including organisms, from penetrating the membrane's surface at any face velocity.)

Table I. Throughput versus membrane* face velocity for a model plugging suspension**

Relative throughputs to membrane Face velocity differential pressures of 2 (gal/min/ft ) IOpsi 50psi

0.5 1.0. 1.0 0.3 1.4 1.4 0.2 1.5 1.4 O.I 1.7 1.5

* membrane=0.45 micrometer polyvinylidene fluoride (Durapore®) * * suspension=yeast extract, bentonite, 12% alcohol ®=reg. trademark ofMillipore Corp., Bedford, MA

Table I contains data for relative throughputs for an 0.45 micron membrane challenged by a model suspension of yeast extract and bentonite. Significant throughput improvements are obtained as the 'face velocity' is decreased. Very similar conclusions were reached by Laufer* who studied membrane throughputs for four wines as a function of face velocity.

A good rule of thumb for final membrane filters is to stay below 112 gallon/minute per square foot of final membrane area. Thus, if the maximum demand ofyour filler is 10 gallons/minute, you need at least 20 square feet of membrane area. At this 'face velocity', the pressure drop at startup will usually be 2 psi or less across the membrane.

For most depth media, including some cartridge prefilters, a similar rule of thumb is 1/8 to 1/4 gallon/minute per square foot of medium. Therefore, the area of pretreatment medium should be sized at two or three times the final membrane area. Proper filter area sizing will give both low startup pressure drops and the most economical filtration costs.

131 If the annual cost of the cartridges approaches the initial capital investment for housings and associated hardware, lower face velocities, i.e., larger membrane area, should be considered. Within a short time, the savings in the disposables will more than offset the investment· in larger housings to hold additional membranes and prefilters.

When sizing a system, there are two other important considerations. The first is the pump or pressure source. For example, if your supplier recommends running the filters (pre and final) up to a differential pressure of 30 psi each, and your filler requires 20 psi operating pressure, this means that a pumping capacity of80 psi (30+30+20) at the filler's demand flow is required in order to continue if both filters are nearly plugged.

Filling with only tank head pressure or with an undersized pump will limit filter throughput by forcing shutdown before acceptable pressure drops have been reached, i.e., before the cartridges are truly plugged.

The second consideration involves sizing a system for throughput. Filler downtime for a larger bottler can be very expensive. If the bottling schedule or beverage variability, i.e., use of the same filters for different beverages, result in unplanned change out downtime, it may be prudent to install duplicate filtration trains (i.e., have the second setup ready to go if the first one plugs). Alternatively, the system should be sized large enough to insure completion of a given batch with plenty of margin for plugging.

DESIGN

Figure 3 shows the proper configuration for all elements in a cartridge filtration system.

"Prefilter" Housing Final Me~brane HOUSing 0 Pressure Gauge ,. Sanitary (Sanitary) .....,. Diaphragm Pump with Sufficient Valve Pressure at Filler ·· Vent .....,. Ball Valve Demand Flow Rate Air/N 2 Vent Supply Welded and From Filling Polished to Tank Filler

Sampling ' Valve Hot Water Access Sampling ' Diffusion Gas Flow And Drain Valve Measurement

Fig. 3. Elements in a cartridge filtration system for aseptic filling.

132 Several important rules must be followed:

1. The final membrane must be housed in equipment designed to minimize both ·holdup and stagnant areas where microorganisms can grow, i.e., no dead ends that cannot be easily sanitized.

2. Sanitary plumbing, including piping, pressure gauges, valves, and connections, must be used for all wines downstream of the final filter.

3. There should be a minimum length of downstream piping and fittings. Pumps should never be placed between the final filter and the filler.

4. Hoses, plastics, and non-polished metals cannot be sanitized easily. Therefore, the final filter housings and all downstream plumbing should be polished stainless steel of grades 304 or 316.

5. Locate valves for sanitary sampling at strategic locations to monitor microbiological counts during filling. These locations should be at points upstream of the filters, between the prefilter and the final filter, and immediately downstream ofthe final filter.

6. Include connections for hot water or steam used to sanitize the system, and for the introduction of pressurized gas to measure filter integrity.

7. Position pressure gauges to monitor the pressure drop (hence plugging rate) of each set of cartridges. It is standard procedure to put pressure gauges immediately upstream and downstream of each housing. If downstream of the final filter, the gauge and connections must be of sanitary design.

8. Pressure relief valves should be included upstream ofboth prefilters and final filters if the pump can exceed the pressure ratings of the housings. These valves also act as vents to permit the escape of entrapped air from the housings (and are commonly located on the top of the housings themselves).

9. Locate the prefilter and the final-filter housings adjacent to each other so that the prefilter effiuent immediately enters the final-filter housing.

There is one very good reason for this proximity requirement; submicron filtration removes many 'protective' colloids, and molecular rearrangements can cause new colloidal aggregates to fonn soon after filtration, sometimes within minutes.

The best examples are the highly-branched beta-glucan molecules which can fonn loose but gummy molecular aggregates with proteins. A tight prefiltration will break up some of the aggregates, but they can reform again within minutes and plug the final filter. Thus, if final membrane filtration follows tight prefiltration and interim storage, the final membrane may

133 become plugged prematurely from new colloids or precipitates induced by the prefilter's clarification and formed during storage.

This same phenomenon is responsible for the occasional clouds, hazes, or precipitates which may appear in the bottle even though the beverage has perfect microbiological stability and was of high optical clarity when bottled. Examples of these precipitates are proteins, protein-tannin " complexes (chill hazes), and tartrates. If a haze or precipitate forms after submicron filtration, additional upstream fining or filtration, enzyme treatment, or cold stabilization may be needed prior to final membrane filtration.

SANITATION

Sanitizing the filters and filling/closure equipment prevents microorganisms from contaminating the beverage after filtration. The source(s) of contamination can be either external or from within the system. Figure 4 shows the most common sources of contamination.

1. Beverage itself 7. Filler heads 2. Plumbing 8. Closures and Conveyors 3. Containers 9. Hopper 4. Conveyors 10. Jaws and Closure equipment 5. Auxilliary fluids 11 . People ,. _Closures 6. Filler bowl 12. Atmosphere • • • 8 2 5 12 9 10 Beverage Filler from Filter 6 Containers 3

Fig. 4. Sources ofmicrobiological contamination.

The best approach is to begin 'thinking sanitary' well upstream of the filter. lftanks, plumbing, and equipment are routinely cleaned and sanitized, the microbiological counts in the beverage arriving at the final filters will be under control.

Many breweries and wineries are exemplary in this matter, and the numbers of microorganisms at all stages of the process are very low. The final membranes are not heavily burdened, and the achievement of a sterile package is more certain.

134 Although the filtrate from the final filter housing is completely free of microbes, this is no guarantee that organisms cannot enter the beverage from contaminated plumbing, filler spouts, containers, closures, the air around the filler, or the hands ofbottling employees.

It is essential that: (a) the entire operation be designed and constructed with attention to sanitary operation, (b) the filling area be located away from possible sources of extraneous contamination, (c) the equipment be sanitized routinely, and (d) equipment, containers and closures be monitored routinely for contamination.

Heat (preferably in the form of hot water) is the most common method used to sanitize a final membrane filter, its housing, and plumbing to the filler, and the filler itself This is because chemicals, even gases, penetrate neither the submicron cracks and crevices which may harbor microorganisms nor the biological surface film often found on internal surfaces of piping and other plumbing attachments.

Suppliers of aseptic filling equipment usually provide complete instructions for heat sanitation, including the recommended frequency. At a minimum, all components should be sanitized once daily before the start of filling. Hot water is preferred, but steam is acceptable.

(If steam is used, even more attention to temperature is required Steam heats by condensing until all surfaces are hot. Only when the temperature of the filler spouts, large metal masses, and gasketing.materials reaches the steam's temperature can the sanitation period commence.)

Table 2. Temperature/time guidelines for equipment sanitation.

Temperature Time

2000f (93°C) 20 minutes 1800f (82°C) 30 minutes 160°F (71 oc) 40 minutes 1400f ( 60°C) 60 minutes

The temperature/time requirements will vary, but Table 2 gives a set of guidelines generally regarded as safe. When filling juices or beverages susceptible to mold-spore contamination, additional measures may be necessary.

All components from the membranes to the filler spouts must be held at the desired temperature for the suggested time. Temperature-sensitive crayons or other temperature indicators can be used to insure that the largest metal mass and most remote locations reach the temperature. The filler spouts should be open to allow water (or steam) to pass through. The times in Table 2 do not include the time to reach temperature.

135 Use carefully filtered water or steam. Dirty water or steam can plug membrane filters as fast or faster than beverages.

Fillers can be sanitized with unfiltered hot water or steam if provisions are made to sanitize the connecting plumbing (e.g., a valve) which separates the filler from the filter during this • process. It must be noted, however, that unfiltered sanitizing water can leave behind inorganic and organic substances which may accumulate in the filler.

When all components downstream of the final membrane have reached the desired temperature, the hot-water flow may be reduced to maintain the temperature. The lower flow will save energy and reduce filter plugging by colloids in the sanitizing water.

After sanitation, the membrane system is brought back to ambient temperature for the integrity measurement, using cold, filtered water.

The procedure for shutdown (end of day) is to chase the remaining beverage with nitrogen, then with cold water, followed by hot water. This will flush the system and may also dissolve colloidal contaminants, such as carbohydrates, that have collected on the membranes' surfaces.

The sanitation procedure should then be repeated. The filtration system will remain sanitary if closed down completely (with valves) overnight or for a weekend.

For long-term storage (more than 60 hours), a sanitizing fluid should be pumped into the system. Acceptable solutions include 0.3% peracetic acid, 25 ppm iodoform, 10 ppm chlorine, and metabisulfite/S02 at about 500 ppm and pH under 5.0. Note: S02 is corrosive to stainless steel.

INTEGRITY TESTING

The integrity test is a method used to check whether there are any larger-than-rated pores or leaks in the system through which a microorganism might pass. Each supplier should provide an integrity test procedure to guarantee their final membrane performance at the rated pore size. These tests can only be done on absolute media, not on nominally-rated prefilters.

Membrane filters should be checked daily before filling to insure their integrity. The test is done after sanitizing the system. It is a good practice to conduct integrity tests at least twice per day, before and after the filling process.

The integrity test measures the passage of gas under controlled conditions through a membrane. There are three popular integrity test methods: bubble-point, diffusional flow, and pressure hold.

1. Bubblepoint: The most popular integrity test is measurement of the membrane's bubblepoint pressure. Because the pores are small, they can be considered as capillaries which hold liquid

136 against applied gas pressure. When the gas reaches a sufficient pressure to overcome the surface-tension forces, it forces the liquid out of the pores and permits the gas to flow through.

Each pore size has a specific gas pressure. The larger the pore, the lower the pressure needed to overcome the surface tension and let the gas pass through (Fig. 5).

Air or N At the bubblepoint Air or N2 2 pressure, the sur­ face-tension forces characteristic of the pore diameter and the fluid are overcome. This allows bulk gas flow through the membrane.

Pressure at Less than bubblepoint bubblepoint pressure pressure

Fig. 5. At the bubblepoint pressure, the surface-tension forces characteristic of the pore diameter and the fluid are overcome. This allows bulk gas flow through the membrane.

The membrane must be completely wet with water at ambient temperature before the test is done. Dry pores permit free gas passage. Because the surface tension of water decreases with increased temperature, inaccurate low bubblepoint pressures are measured at higher temperatures. For example, at 60°C compared to 20°C, the surface tension of water is lower by almost 10%.

A cylinder of nitrogen or compressed air, (filtered if possible), a pressure regulator, and an accurate pressure gauge (readable to 1 psi increments) are required to do the test. Carbon dioxide cannot be used because of its relatively high solubility in water.

Gas is introduced into the system upstream of the membranes. It is first applied at low pressure (about 5 psi) to force the upstream water through the pores oft~e filter. The bubblepoint pressure is determined by increasing the gas pressure slowly over a period of three to five minutes until a continuous flow of large bubbles appears downstream.

137 The bubbles may be observed using an in-line site glass, or a piece of tubing may be aseptically attached downstream of the housing with the other end immersed in a container of clean water to visualize the appearance of the bubbles.

For an 0.45 micron membrane, the bubblepoint is usually greater than 20 psi. lflarge bubbles appear downstream at a lower pressure than that set by the supplier, the membrane fails integrity.

2. Diffusional flow: A small stream of very tiny bubbles may appear below the bubblepoint pressure. This is caused by gas molecules dissolving into the water and then diffusing through the wetted membrane matrix. At atmospheric pressure downstream of the membrane, the gas reforms as tiny bubbles.

When large membrane areas are used, the diffusional passage of gas may be considerable, and it may be impossible to clearly distinguish between diffusional flow and the bubblepoint.

The diffusional flow integrity test involves measuring this gas diffusion rate at a pressure less than (typically 80% of) the bubblepoint pressure. For membrane areas over about 100 square feet, the diffusional test is the most reliable method.

When doing this type of integrity test, an accurate method of measuring gas flow is needed such as a flow meter or a piece of tubing to convey the gas into an inverted water-tilled graduated cylinder. The rate of water displacement in the cylinder is the diffusional flow.

Manufacturers will specify a minimum acceptable diffusional flow rate at (before) which the membrane's integrity is judged unacceptable.

3. Pressure hold: The third integrity test is the pressure hold, where the filters are pressurized to a preset level, typically 80% of the bubblepoint pressure. The gas supply is then turned off, and integrity is determined by how fast the pressure drops.

A typical specification for acceptance (depending upon the upstream gas volume) may be that the pressure drops no faster than 2 psi in five minutes. A faster drop indicates that openings exist through which the gas can pass, and that these openings are larger than the membrane's rated pore size. This method is similar to the diffusion test, but not as accurate.

4. Troubleshooting: There may be several reasons for membranes to fail the integrity test that are not related to true pore integrity. For instance, the membrane may not be completely wetted; some of the pores may not be tilled with water.

Sometimes the membrane is not at fault at all; most systems include o-rings or gasket-type seals which may be the cause for integrity failure. However, the net result is the same; the integrity of the system is not sufficient to maintain a microbiologically stable filtrate, and filling should not commence until the problem is found and corrected.

138 After sanitation, it is possible to visually check the cartridges and seals by removing the bowl (or dome) of the final membrane housing without contaminating the downstream areas. If any seals are interrupted, however, the sanitation procedure must be repeated.

AUXILIARY FLUIDS, EQUIPMENT AND PEOPLE

There is no substitute for sanitary operation of the filler and closure equipment. Auxiliary fluids, the containers, the closures, and people are all possible sources of contamination.

Gases for stirrup-lifting, purging, counter-pressure at the filler, conveying, and jetting (blasting foam offbeer after it's in the can) must be sterile-filtered with membrane filters capable of being sanitized (preferably by steam), and integrity-tested. The plumbing which delivers the fluids, and all surfaces with potential fluid or closure contact, must also be kept clean and sanitized regularly.

Any openings between the filter and the filler, such as sampling valves, must be flamed or sprayed with 90% isopropanol or 70% ethanol before and after opening.

Corks, the corker surfaces, the hopper, the corker vacuum assemblies, and especially the corker jaws are notorious havens for organisms. If there is any question about contamination, soak the corks in 500 ppm metabisulfite at a pH below 5.0 for 24 hours before use.

Corker jaws may be kept hot to prevent contamination. Otherwise, they should be cleaned and sanitized on the same schedule as the filters and the filler, and maybe more often. Some wineries spray corker jaws at every break and after any maintenance contact with 70% ethanol.

New bottles are usually free of contamination. They should be stored in unopened and undamaged packages or on sealed pallets in a clean, dry area. If exposed to moisture/humidity or if stored for a long time, check for dust, bugs and sterility before use.

Heat sanitation of the filler spouts is the only proven method of insuring freedom from contamination. This is best accomplished by allowing a trickle flow of hot water through each spout as prescribed in Table 2. ·

An additional insurance during filler downtime is to spray a stream of appropriate sanitizing fluid , for example 25 ppm iodoform or 90% isopropanol (or 70% ethanol) onto each spout. Frequent disassembly and cleaning of each spout, albeit time-consuming, represents the ounce of prevention worth a pound of cure later.

Open windows or doors, fans, fork-truck movement, overhead bird or rodent or bug drippings, etc., can all bring unwanted contamination to the filling and closure areas. All air movements should be controlled by minimizing traffic. The filler and closure equipment should ideally be isolated even to the point of installing clean low ceilings and sidewalls.

139 Finally, attention p"aid to personal hygiene by employees working around the filler, containers, closures, and conveying devices is essentiaL Operators should wear masks, disposable sterile gloves, clean clothing, and even protective footwear.

Once an unwanted organism from any source invades a piece of equipment, the only solution is complete isolation, cleaning and sanitation, and renewed attention to all auxiliary fluids, surfaces, containers, and personneL

Serial numbering of cases, or .at least pallets, allows contaminated product to be identified and isolated for reprocessing, thus avoiding having to reprocess the entire lot or the whole day's bottling.

MONITORING

Monitoring the equipment and the product is important to ensure microbiological stability. Sanitary sampling valves should be used to collect in-line product samples. Some suppliers provide kits to monitor the filler spouts, corker heads, bottles, closures, and even the air around the filler. Swab tests are an easy way to check-up on the efficiency of your sanitation procedures.

To test samples for the absence of spoilage organisms, you will need a vacuum source and flasks, petri dishes, lab-sized membranes, nutrient media, an incubator, forceps, a flame burner, and a magnifying viewer or microscope (if possible )(Fig. 6). Samples can be sent out to an outside lab for testing.

Initial, mid-run, and end-run produce should be sampled, and both filler spouts and corker heads should be monitored daily (or at least weekly) with a Millipore Swab Test Kit, for example. By catching a problem early, it may be remedied so that contaminated product is minimized.

Good housekeeping, strict adherence to a schedule or a routine, and the use of proper sanitary procedures are more important than fancy equipment for proper monitoring.

140 Equipment/supplies required for micro­ biological monitoring: (A) sanitary sam­ pling valve: (B) collection container; (C) gridded membranes; (D) forceps; (E) fil­ ter holder or funnel; (F) vacuum source; (G) petri dishes; (H) media: (I) incubetor; (J) magnifying lens or microscope; (K) swabs and samplers.

~------I F

K 8 ...... ___,. <.t~<.q;?j( c

Fig. 6 Equipment/supplies required for microbiological monitoring: A) sanitary sampling valve~ B) collection container~ C) gridded membranes~ D) forceps~ E) filter holder or funnel~ · F) vacuum source~ G) petri dishes~ H) media~ I) incubator~ J) magnifYing lens or microscope~ K) swabs and samplers.

SUMMARY

Membranes have been successfully employed to give both ultimate clarity and stability since the late 1960's_ With proper design, attention to sanitation, routine integrity testing and monitoring, this technology can be easily managed.

Before buying expensive housings, filter media and monitoring equipment, it's best to consult with reputable suppliers ofboth membrane filters and monitoring equipment Their experience can help you establish both economical and proven performance in aseptic filling.

141 USE OF THE WINE AROMA WHEEL

John Buechsenstein McDowell Valley Vineyard, Hopeland, CA

Sensory evaluation (of wine) may be defined as the ,.measurement, quantification, and 11 interpretation of characteristics of wine perceived by smell, touch, taste, and sight • (Noble lecture notes). When we evaluate wine, perhaps as much as 70% of what we perceive is due to our sense of smell. As humans, especially if we've been brought up in a culture where wine is not a normal, regular part of our cuisine (the majority of us in America), we often have an underdeveloped ability to describe what we smell.

Our sense of smell is connected to the part of our brain dealing with emotional responses, by-passing our intellectual or analytical evaluation. As a result, our first response, when asked 11 11 11 to describe a wine, will be almost certainly hedonic: ''Yum! or Yuk! • Even as a scientist, normally used to responding in an analytical manner, if he or she is a novice wine taster, will lack the descriptive tools needed to describe a wine and lapse into a ,.gut,. reaction. According to Dr. Jim Lapsley at U.C. Davis: ,.A scientist, when asked to rate forty wines into 11 bad, good, or great .. , said, 'I'm never laconic, in matters hedonic, since I know what I lave and I hate!"' (Lapsley, 1990)

With a bit more experience and a natural poetic tendency the same person, while believing he is being more precise, will offer an evaluation such as 11 an amusing wine with playful aromas and velvety flavors,..

It is difficult to use our descriptive vocabulary precisely, especially without some training as to what to expect of a certain wine type. Training and experience help us develop a context for different wine types and styles. The question can be asked: Do we want to develop our descriptive ability in this manner?

YES! Whether we are just consumers of wine, sellers of wine (off or on-premise), or winemakers and winegrowers, or all of the above, much of our enjoyment can come from talking about wine. Much the same as our enjoyment of art or music can be enhanced by at least a modicum of study our enjoyment offood (wine) can also result from knowing what goes into it and by having the vocabulary to express it to others at table.

Psychologists have identified a human expressive difficulty called the 11 tip of the tongue,. phenomenon. This refers to our inability to connect (among other things) sensory input with our intellectual brain center in order to speak in descriptive terms; i.e., we seem to know what it is we are smelling or tasting but the word necessary to describe it is right on the tip of our tongues. Training (wine-sensory exercises) can help us overcome this barrier and at every level of wine appreciation can make and communication less intimidating and more enjoyable.

142 Figure 1. Modified ASEV Wine Aroma Wheel showing first-, second-, and third-tier terms. Noble et al., (Am. J. Enol. Vitic. Vol. 38, No.2, 1987, pg. 143).

For this reason a system of wine aroma terminology was developed and graphically represented in a useful format known as the Wine Aroma Wheel (See Fig. 1).

I'd like to introduce the Aroma Wheel to you today and briefly show, with some wind-odor examples, how it can be useful in training both novice and professional to develop a meaningful descriptive vocabulary for wine. More than simply supplying adjectives the Aroma Wheel can be used to train individual to smell for specific categories.

The Aroma Wheel is arranged by displaying three levels or tiers of descriptive specificity. The most general, or first-tier terms are in the inner circle. These help us identify the broad category of smell (see Fig. 2).

143 Third Tier .-.

Second Tier .-.

FirstTier ~

GREEN BEANS

Fig. 2. Spicy, fiuity, and vegetative sections ofthe Wine Aroma Wheel, Noble et al. (Am. J. Enol. Vitic., Vol. 38, No.2, 1987, pg. 143).

The second-tier represents more specific subdivisions of the first-tier terms. For example, the first-tier term "fiuity" is divided into several groups which narrow down the category offiuit that the odor suggests. First and second-tier terms are good general descriptors when the taster perceived general, non-specific odors pertaining, in this case, to a fiuit origin.

The t.hird-tier presents the most specific descriptors. If a particular fiuity odor is present at a high enough level, it may be possible to use one of these terms. All of the third-tier descriptors on the Aroma Wheel can be defined by preparing and providing reference standards specified in the cited AJEV paper (Noble, et al., Table 1). Quite useful is the fact that most substances needed for reference preparation may be easily obtained, usually from a grocery or natural foods store. Some of the more exotic can be found in lab supply catalogues available to wineries.

144 In a more rigorous setting, such as a winery or a wine flavor research lab, Descriptive Analysis using the Aroma Wheel terms might take on a more formal definition: "a process of describing and quantifYing the perceived sensory characteristics of(a wine)" (Noble, lecture notes). In this case a group of selected panelists would, under the direction of the researcher> agree on which meaningful descriptors to use for a particular wine type > train their ability to recognize and quantifY each descriptor using prepared reference standards > develop a score card to use in rating the intensity of each perceived odor. It may be necessary to screen out which could influence the panelists' decisions using opaque black glasses or tasting booths with red lighting.

Results can be displayed graphically using a circular "polar-coordinate" graph, sometimes called a "radar-graph" or a "spider web". Each radius represents a particular descriptor with zero intensity at the point of origin and increasing intensity moving out toward the circumference. (Several slide examples of polar graphs were shown.) Such graphs can be combined or superimposed to look for differences between wines or experimental treatments of a particular wme.

With little practice you too can bridge the gap between knee-jerk, hedonic wine evaluation and precise aroma/flavor description. Consider the following quote from Lewis Carroll: Alice in Wonderland on drinking the bottle labeled "DRINK ME" said "It had a mix-up flavour of cherry­ tart, custard, pineapple, roast turkey, toffee, and hot-buttered toast", to which Dr. Jim Lapsley quipped, "Was it over-oaked Chardonnay?" (Noble, lecture notes).

A flight of eight unidentified wine aroma reference standards will be passed around. Please smell them and write down the descriptor from the Aroma Wheel that best describes each (See Fig. 3).

Aroma Exercise: Refer to the Aroma Wheel in your handout and find the descriptor that best matches each odor reference. Following is the correct list:

First Tier Second Tier Third Tier

A oxidized oxidized aldehydic B earthy moldy corky c floral floral geranium tone D chemical pungent ethyl acetate E floral floral linalool F vegetative canned/cooked asparagus G fruity berry raspberry H vegetative fresh eucalyptus

Fig. 3 Key to aroma reference standards distributed to audience.

145 The Wine Aroma Wheel is a valuable tool that can be used to train novice and professional tasters to develop a precise descriptive vocabulary for wine. Thanks very much for your attendance and participation.

References

Lapsley, James. 1990. Lecture Notes, Dept. of Viticulture & Enology, U.C. Davis.

Noble, Ann. 1988. Lecture Notes, Dept. ofViticulture & Enology, U.C. Davis.

Noble, A.C. et al. 1987. Modification of a standardized system ofwine aroma terminology. Am. J. Enol. Vitic. 38(2):143-146.

146 LAKE ERIE QUALITY WINE ALLIANCE--ITS MISSION AND PRESENT STATUS

Bob Mazza, President Mazza Vineyards, North East, PA

A. What Is the Lake Erie Wine Alliance?

The Lake Erie Quality Wine Alliance is an organized group of wineries located within the Lake Erie Appellation ofNew York, Pennsylvania and Ohio. The Alliance's main goal is to promote the region as a cohesive viticultural area producing quality wines.

The mission of the Lake Erie Quality Wine Alliance is to ensure that the wines of the Lake Erie Appellation be recognized as a quality product. The Alliance believes that this can be accomplished through the development of marketing programs that enhance the sales and profits ofLake Erie wines through retail and wholesale efforts.

The Lake Erie Quality Wine Alliance believe that we can relay upon the quality of the wines of our region to promote the quality image of the Lake Erie viticulture region. In addition, the Alliance will promote a consistent quality image for the wine growers in the Lake Erie Region.

The strategic plan for the Lake Erie Quality Wine Alliance includes:

a. identifying wines that promote a quality Lake Erie image. b. developing a marketing program to enhance the sale ofLake Erie wines through wholesale and retail efforts. c. creating public relations and advertising campaigns to promote the region and member wineries.

B. Who are the Members of the Lake Erie Quality Wine Alliance?

A requirement for membership is that the winery be located within the Lake Erie viticultural region. Members as of January 1, 1994, include the following:

Ohio Buccia Vineyards, Chalet Debonne Vineyards, Firelands Winery, Klingshim Winery, Lonz Winery, Markko Vineyards, Mon Ami Champagne Company, Old Firehouse Winery. Pennsylvania Conneaut Cellars Winery, Mazza Vineyards, Penn Shore Vineyards, Presque Isle Wine Cellars.

C. History of the Lake Erie Appellation District

The Lake Erie viticulture area has a 150-year history of grape growing and winemaking. There are approximately 40,000 acres of commercial vineyards in the Lake Erie viticultural area,

0 147 located in the states of New York, Pennsylvania, and Ohio.

The Lake Erie viticultural area was established as an Appellation District in November 1983. It is distinguished from the surrounding areas by its nearness to the lake, which exerts a moderating influence on the area. The influence of Lake Erie on the climate is the fundamental factor that permits viticulture in this area. There are presently two approved viticultural areas located within the boundaries of the Lake Erie viticultural area: Isle St. George and Grand River Valley.

D. Member Survey

One of the first steps undertaken by the new Lake Erie Quality Wine Alliance was to determine exactly what each member and non-member winery considers to be the quality wines of this region. Therefore, a comprehensive survey was developed as a means for helping to chart the course in the search of an identity as well as an image of quality for Lake Erie wines.

Of particular interest to this group was the information gathered on recent sales trends. Approximately 20 I, 000 gallons of wine were reported sold in 1992, with the varieties below comprising the following percentages of sales:

Catawba, pink and white 19% Chardonnay 14% Concord 13% Vidal 10% J. Riesling 9% Niagara 9% Seyval 5% Cabemet Sauvignon 4% Delaware 3% or less DeChaunac " Chancellor " Steuben " Vignoles " Pinot Noir " Chambourcin " Pinot Gris " Blush " " Haute Sauterne "

148 E~ Future Plans of the Lake Erie Quality Wine Alliance

The Alliance has a number of plans for 1994. Immediate plans call for the development of a wine tour brochure for informational and marketing purposes. Special events such as media functions, dinners and wine tastings will also be undertaken, along with continued involvement with wine educators through the upcoming Wine Educators Conference in Toronto. In addition, the Alliance seeks to expand its membership into New York state and throughout the Ohio Lake Erie region. As a service to member wineries, the Alliance will institute a Grape Clearinghouse Hotline later this year. The hotline will alert members as to which specific varieties each winery has a need for or abundance of Non-members will be charged a nominal feel for use of this servtce.

149 CARNEROS QUALITY ALLIANCE--THE DEVELOPMENT OF AN APPELLATION

Eugenia Keegan, CEO/President Bouchaine Vineyards, Napa, CA

For decades it has been known that the Cameros microclimate is particularly well suited for the cultivation ofPinot Noir grapes. The question still remained, however, whether or not there are characteristics in Pinot Noir wines made from Cameros grapes that are common to all Cameros Pinot Noirs, but are not generally found, or not found to be as intense in Pinot Noir wines made from grapes in other regions. In other words, is there in fact a recognizable Cameros style ofPinot Noir?

In 1986, the Cameros Quality Alliance set out to answer this question. In order to conduct a scientifically valid experiment, one in which the results could be defended and supported with research methods of the highest credibility and standards, the Alliance contacted Dr. Ann Noble of the Department of Viticulture and Enology at the University of California, Davis. The experiment was conducted, under her guidance, by two UC Davis students, one a PhD candidate and the other a Masters student.

A tasting was designed to compare Cameros wines with wines from both Napa County (non-Cameros) and Sonoma County (non-Cameros). Wines from two vintages (1981 and 1983) were tasted in order to minimize any differences due to vintage. A panel of twelve trained judges tasted a total of28 wines (10 Cameros, 9 Napa, 9 Sonoma) twice each in 14 sessions held on different days. All wines were tasted blind, four wines per session and no wine was presented in combination with any other wine more than once. The judges were asked to score each of the attributes chosen to describe Pinot Noir wine (fresh berry, berry jam, spicy, mint, prune, vegetal, smoke/tar and leather) on a linear scale of 1 to 10.

After the wines had been tasted twice by all judges, the results were correlated and subjected to a number of complex statistical analyses. The results were then displayed on a two­ dimensional graph which showed 9 ofthe 10 Cameros wines clustered together in one area, representing high cherry, fresh berry, spicy and berry jam intensities. The wines from Napa and Sonoma Counties were not clustered with the Cameros wines or with themselves; they were scattered all over the graph.

Chemical analysis of all the wines was done and the results showed that the Cameros wines again clustered together, away from the other wines, with regard to the free and bound so2 levels. All ten of the Cameros Pinot Noir wines had S02 levels which were significantly lower than the wines from the other regions.

This study has proven that there are characteristics--namely fresh berry, berry jam, spicy and cherry--which are consistent among Cameros Pinot Noirs and which are significantly more intense than in Pinot Noirs from other regions.

150 Officially recognized as a winegrowing appellation in 1983, the Cameros region is celebrating its tenth anniversary. Unlike many grape-growing areas whose boundaries follow political lines, Cameros was established strictly according to microclimatic influences.

Today the region, spanning the southern ends of Napa and Sonoma Counties, is recognized for producing distinctive Pinot Noir, Chardonnay and other varieties as well as sparkling wines. The appellation's bay-influenced climate, low rainfall and shallow soils all contribute to the unique properties of grapes produced here.

In 1992, Highway 121 between Highway 29 in Napa and Highway 37 in Sonoma County was renamed the Cameros Highway by the California legislature, making it the first highway named after a winegrowing region.

Long an important route, the Cameros Highway was first travelled in the 1800's. Drawn to the Cameros region for its moderate climate, settlers came to the area to raise sheep and cattle, as well as grapes and other produce. In fact the region takes its name from the Spanish word for sheep--Cameros.

The first Cameros vineyards were planted in the 1830's and wine grapes and wineries are the primary agricultural focus now. More than 6,500 acres of grapes dot the hillsides and 26 wineries. belong to the region's trade association--The Cameros Quality Alliance.

The CQA is an alliance of growers and vintners in the Cameros appellation. CQA winery members include:

Acacia Kent Rasmussen Winery Beaulieu Vineyard Louis Martini Winery Bouchaine Vineyards McKenzie-Mueller Vineyards Buena Vista Winery Mont St. John Cameros Alambic Distillery Ravenswood Cameros Creek Winery Robert Mondavi Winery du Val Robert Sinskey Vineyards Clos Pegase Roche Winery Codorniu Napa Sainstbury Domaine Cameros Schug Cameros Estate Domain~ Chandon Sonoma Creek Winery Gloria Ferrer Truchard Vineyards Havens Wine Cellars ZD Wines

151 HISTORY OF THE OHIO WINE PRODUCERS ASSOCIATION MEMBERS AND THEIR ROLE IN THE OHIO WINE INDUSTRY

1895 Steuk's Wine Co. Started bonding nwnber 1937 Ferrante Winery Original winery founded 1947 Lonz Winery Middle Bass hangover 1%1 Art Vindetti Concord Wine 1%2 Born into Ohio wine family 1%5 Philip Wagner referred me to Dr. Frank 1967 Dr. Frank visit 1970 OWPA First ·short course 1971 Debonne Winery Business started 1973 Klingshim Winery AI Klingshim attended 3rd short course and became a member of OWPA soon after 1973 Visited Markko; Planned my own vineyard 1974 Meeting with OSU staff on forming OWPA 1976 Meiers Winery Started in Ohio wine business by purchasing Meiers 1977 Tom Johnson is given Ch. Lagnap - 3 days old - Seyval Blanc -out of the vat -at Restaurant by Dr. Tom Wycolt. Tom survived. 1977 Visited Dr. Frank. Gave us permission to grow vinifera at Firelands & Meiers Meeting with family- What Donnie should do instead of teaching. Tony said to do something for the wine business. 1978 Made K.W., Inc. full time winery 1979 House Bill 302 1979 Ferrante Winery New location in Geneva 1979 First Association with Ohio Wine Industry and First Taste River Rouge/H.B. 302/Bruce Benedict 1981 Ohio Grape Industries Committee created 1983 Spent time in Germany Riesling 1984 Wyandotte Butler's bought Wyandotte 1985 Graduated - full time at the winery 1986 The Unicord Motorcycle Club invaded Chalet Debonne in search of more classic white, conswned at Brennan's Restaurant. Mistaken by Russ for Hell's Angels 1987 Mary McNellie pours Fireland's Chardonnay at Galantine which becomes one of Colwnbus' first house white wine by the glass 1988 Moved to Ohio from Boston and drank my first Ohio wine - a Debonne Niagara. My wife loved it and we left a card to buy the shop 1989 Ferrante Winery Ferrante Wine Farm Inc.- full service restaurant 1990 William Graystone Opened William Graystone 1990 Purchased small beverage shop in Madison named "Wet Your Whistle" 1993 Best of Show in San Francisco with 1992 JR Lake Erie 1993 Lake Erie Wine Alliance 1993 First Double Gold in California- Native Ohio Grape Variety 1993 Steuk Inc. 1994 OWPA Short Course

152 SUMMARY OF THE RESULTS OF THE S.W.O.T. ANALYSIS

This list was compiled according to the 3 "dots" which people were asked to place next to the items they felt were most important. They are listed beginning with the item which received the highest number of dots down to the item which received the lowest number of dots (or votes).

Strengths

1. Excellent fruit growing area 2. Geographical location, 500 miles of2/3 ofU.S. population= large untapped consumer market. 3. Strong desire for winery owners to succeed 4. Good cooperation with OARDC and OSU 5. Support ofMeier's- big operation has helped the little guys 6. Personality and family-owned nature are appealing to consumers 7. Quality price, good value 8. Aggressive cooks and chefs potentially supporting innovations of the industry 9. Wine is food; strong agricultural market 10. Support of state administration Dept. ofDevelopment, governor's and Department of Agriculture

Weaknesses

1. Image - sweet 2. Non-promotion of our wine in national media and competitions 3. Retailer reluctance 4. inconsistent quality 5. Ohio not perceived as a wine state 6. Packaging 7. Inferiority complex 8. Undercapitalized 9. Focus on proper varieties (have not identified competitive varieties) 10. Lack of educated consumers 11. Interstate commerce 12. Lack of promotion with other national tourist and other allies 13. 3-Tier system - minimum pricing

153 Opportunities

1. Better media contacts 2. Work together for quality 3. Industry is still evolving - change to grow positively 4. Wine different from other alcohol containing beverages 5. Room to educate consumers 6. Regionalism is key to marketing 7. Strive to make a better produce/improve 8. Health benefits of wine 9. Untapped markets, such as restaurants and upscale stores 10. Maximize public tasting opportunities, i.e., festivals 11. Honest evaluations of wines 12. Tourism growing in Ohio

Threats

1. Laws - UPS restraints, tasting and sampling restraints (shipping in general) 2. Taxes! (consumption) 3. "Neoprohibitionists" as an organized group (C.S.I.P.) 4. Alcoholism - public's perception 5. Inconsistent quality 6. Weather 7. Aging consumer base 8. Complacency 9. GATT and other trade agreements 10. Regulations

154 1994 GRAPE-WINE SHORT COURSE SPEAKERS & PARTICIPANTS

TOASTMASTER: Dr. Garth Cahoon KEYNOTE SPEAKER: Dr. Thomas Payne

SPEAKERS:

Kenneth Bement, Owner Wet Your Whistle Wine Shop Madison, OH

Bruce Bordelon, Assistant Professor Department ofHorticulture Purdue University West Lafayette, IN

John Buechsenstein, Winemaker McDowell Valley Vineyards Hopland, CA

Garth Cahoon, Professor Emeritus Department ofHorticulture OARDC/OSU Wooster, OH

Mark Chaffin, Field Technical Serv. Mgr. Diversey Crop E. Sandwich, MA

Mike Ellis, Professor Department ofPlant Pathology OARDC Wooster, OH

ArnulfEsterer, Owner Markko Vineyard Conneaut, OH

Nick Ferrante, General Manager Ferrante Winery Geneva, OH

Dave Ferree, Professor Department ofHorticulture OARDC Wooster, OH

155 SPEAKERS:

Kent Glaus, Partner Portage Hills Vineyards Suffield, OH

Douglas Grubler, Professor Department ofPlant Pathology University of California Davis, CA

Ellen Harkness, Microbiologist Department ofFood Science Purdue University West Lafayette, IN

Eckhard Kaesekamp, President Euro Nursery & Vineyard, Inc. Jordan, Canada

Eugenia Keegan, CEO/President Bouchaine Vineyards Napa, CA

Robert Kirtland, Wine Educator Toledo Blade Toledo, OH

Bob Mazza, President Mazza Vineyards & Lake Erie Quality Wine Alliance North East, PA

Peter Meier, US Marketing Mgr. Process Food & Beverage Filtration Millipore Corp Bedford, MA

Mark Meyer, Director Food & Beverage Quail Hollow Resort Concord, OH

156 SPEAKERS:

Diane Miller, Associate Professor Department ofHorticulture OARDC Wooster, OH

Thomas Payne, Director OARDC/OSU Wooster, OH

Roland Riesen, Res. Enologist Department of Horticulture OARDC Wooster, OH

Tim Rios, Exe. Chief Quail Hollow Resort Concord, OH

Karla Roehrig, Associate Professor Department ofFood Science & Technology The Ohio State University Columbus, OH

Mike Shank, Club Mgr. Catawba Island Club Port Clinton, OH

Marta Stone LEADERship Ashtabula Co. Rock Creek, OH

Christian Syberg, Winemaker Meier's Wine Cellars, Inc. Cincinnati, OH

Roger Williams, Professor Department ofEntomology OARDC Wooster, OH

Donniella Winchell, Exe. Director Ohio Wine Producers Association Austinburg, OH

157 SPEAKERS:

Andy Wineberg, Owner The Winery at Wolf Creek Norton, OH

REGISTRANTS:

Bruce Benedict Ohio Dept. of Agriculture 65 S. Front St. Columbus, OH 43215

Dalton Bixler Breitenbach Winery 5934 Old St. Rd. 39 Dover, OH 44622

Robert Boas 2376 Beechwood Blvd. Westlake, OH 44145

Marc Boettcher Presque Isle Winery 9440 Buffalo Rd. North East, PA 16428

Tom Bowen 1366 N. Abbe Rd. Elyria, OH 4403 5

Done Bower DLB Vineyards 30311 Clemens Rd. Westlake, OH 44145

Lawrence Brett Shade Tree Winery 206 W. Nash Wilson, NC 27893

158 REGISTRANTS:

Fred Bucci Bucci Vineyards 518 Gore Rd. Conneaut, OH 44030

.. Bill & Jane Butler 4640 Wyandotte Rd. Columbus, OH 43230

John Christ Vineyards 32421 Walker Rd. Avon Lake, OH 44012

John Christensen 1112 E. Cooke Rd. Columbus, OH 43224

William Croxton Briar Patch Nursery POBox 34407 Pensacola, FL 32507

Daniel C. Richard Daniel Vineyard Box 888 Beckley, WV 25801

George Danko Dankorona Winery 155 Treat Rd. Aurora, OH 44202

TonyDebevc Chalet Debonne 7743 Doty Rd. Madison, OH 44057

David Dexter RD #3, Box 2138 Bristol, VT 05443

159 REGISTRANTS:

Decesare Hill Interlikn America, Ltd. 106 E. Market St. Warren, OH 44481

Eckhard Kaesekamp • Euro Nursery 3197 Culp Rd. Jordon, Ontario, Canada LOR ISO

Harry & Karen Ewing 6170 Clum Rd. #2 Harrod, OH 45850

Sonia Fails Lake Erie Quality Alliance PO Box 11755 Eria, PA 16514

Larry J. Adams POBox 1532 Marion, OH 43302

Nick Ferrante Ferrante Winery 5585 St. Rt. 307 Geneva, OH 44041

David & Nancy Genger 3319 Stockholm Rd. Cleveland, OH 44120

Roger & Ann Gossett Fox Hill Vineyard POBox404 Steubenville, OH 44120

Hiram Hardesty 3310 Warrensville Center Rd. Cleveland, OH 44122

160 REGISTRANTS:

Russell Harding 187 Noroton Ave. Darien, CT 06820

C.A. Harris R.avenhurst Cellars P0Box6 Mt. Victory, OH 43340

Richard Raynor National Grape Co-op POBox 508 Geneva, OH 44041

John Hempstead 1090 Co. Rd. #4 Bellefontaine, Oh 43311

JeffHochschild 2045 Stumpville Jefferson, OH 44047

Robert Rozak Clinton, PA 15026

Jim Iubelt 10945 Burlington Ridge Chardon, OH 44014

Susan Jeager Susan Jeager Graphic Designs 6030 Laskey Rd. Hartsgrove, OH 44085

John Christ John Christ Winery 32421 Walker D. Avon Lake, OH 44012

Tom Johnson 1330 West Blvd 624C Cleveland, OH 441 02

161 REGISTRANTS:

Joseph Kaschalk 5737 S. Park Parma, OH 44134

Sharon Klay Fayette Spring Farm 111 Riding Trail Pittsburgh, PA 15252

Lee Klingshirn Klingshirn Winery 33050 Webber Rd. Avon Lake, Oh 44012

Kovacic 4018 Middle Ridge Rd. Perry, OH 44081

Charles Lavicka 5299 E. 96th St Garfield Heights, OH 44125

Dean Ley R14659 St. Rt. 116 Van Wert, OH 45891

Arnulf Esterer Markko Vineyard RD #2, S. Ridge Rd. Conneaut, Oh 44030

Shirley Martin Country Wines 333 Babcock Blvd. Pittsburgh, P A 1523 7

Theresa M. Luce 2045 Stumpville Rd. Jefferson, OH 44047 ..

162 REGISTRANTS:

Art McGlaughlin Dover Vineyards 24945 Detroit Rd. Westlake, OH 44145

Dave O'Brien 475 Harvey Kent, OH 44240

Dave Otto Old Firehouse Winery PO Box 310 Geneva-on-the-Lake, OH 44041

Robert Gottesman Paramount Distillers 3116 Berea Rd. Cleveland, OH 44111

Art Pietrzyk St. Joseph Vineyards 6060 Madison Rd. Thompson, OH 44086

Tom & Mary Quilter Shamrock Vineyards Box 11 C H#25 Waldo, OH 43356

Dave Rechsteiner Willow Hill Vineyards 5460 Loudon St. Johnston, OH 43031

Fred Roehrig 4800 Hayden Blvd. Columbus, OH 43221

James Roush 4742 W. 32nd St. Cleveland, OH 44109

163 REGISTRANTS:

Claude Rousseau 124 N. 33rd St. Newark, OH 43055

Louis Schiappa 419 Dresden Ave. • Steubenville, OH 43952

David Scholls 46 Co. Rd. 26 Marengo, OH 43334

Ken Schuchter Valley Vineyards 2276 East US 22-3 Morrow, OH 45152

Terry Setterlin 5560 Shnintzinger Rd. Hillard, OH 43026

Larry Sprigg Steuk Winery 1001 Fremont Ave. Sandusky, OH 44870

John Stephens 8437 Settlers Passage Brecksville, OH 44141

William Sturkey Ohio Dept. of Agriculture 4642 N. High St. Columbus, OH 43214

Tom Swank Spring Hill Orchards 2000 Van Pelt Rd. Geneva, OH 44041

164 REGISTRANTS:

Christian Syberg Meier's Wine Cellars 4502 Hunt Rd. Blue Ash, OH 45242

,. Gary Vest Remsen Valley Farms 2395 Remsen Rd. Medina, OH 44130

AI Widiger 12961 W. Lenden Lane Parma, OH 44130

Mark & Lin Wilson 31 E. Pacemont Rd. Columbus, OH 43202

Andy & Hart Wineberg Wolf Creek Vineyards 2637 S. Cleve-Mass Rd. Norton, OH 44203

Dick Woodworth 6501 Middle Ridge Rd. Madison, OH 44057

LeeWyse Rainbow Hills Vineyards 26349 TR251 Newcomerstown, OH 43832

Ben Sparks 8310 N. Possum Trot Rd. Unionville, OH 47468

• Ravenhurst Champagne Cellars POBox6 Mt. Victory, OH 43340 • Edmund L. Wagoner 945 Satin Rd. Columbus, OH 43204

165 TRADE SHOW PARTICIPANTS

George F. Ackerman Co. PO Box 157, 300 Mill St. Curtice, OH 43412

Cliff Brooks Photography 113 Forest Hill Dr. • Avon Lake, OH 44012

Wine Bottle & Packaging, Inc. 100 Jutland Rd. Etobicoke, Ontario, Canada M8Z 2H1

Bureau of Alcohol, Tobacco & Firearms Plaza South One, Suite 11 0 7251 Engle Rd. Middleburg Heights, OH 44130

Country Wines 3333 Babcock Blvd. Pittsburgh, PA 15237

Canton Cooperage Co. POBox 548 , KY 40083

Anak Int'l Corp. POBox 8945 Cincinnati, OH 45208

Euro Nursery & Vineyard, Inc. 3197 Culp Rd. Jordon, Ontario, Canada LDR 1SO

Angle Cloth 28 School St. Stony Creek, CT • Elaine& Co. 18 W. Ashtabula St. Jefferson, OH 44047 •

166 TRADE SHOW PARTICIPANTS

Prospero Equipment Corp. 134 Marble Ave. Pleasantville, NY I 0570

Elf Atochem North America, Inc. 6367 Rider Rd. Reynoldsburg, OH 43068

Criveller Co. 6935 Oakwood Rd. Niagara Falls, Ontario, Canada

Scott Laboratories, Inc. PO Box 4559 Petaluma, CA 94955

Presque Isle Wine Cellars 9440 Buffalo Rd. North East, PA 16428

Ciba Plant Protection PO Box 391 Wellington, OH 44090

APM, Inc. 7355 Trans Canada Hgwy #220 Montreal, Quebec, Canada H4 T I T3

•

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- THE SPEAKERS *******************

From top to bottom Peter Meier Millipore Corporation Karla Roehrig Ohio State University Bruce Bordelon Purdue University

.. .J

From top to bottom From top to bottom Douglas Gubler Dave Ferree University of California, Davis Ohio State University/OARDC John Buechsenstein Diane Miller McDowell Valley Vineyards Ohio State University/OARDC Mike Ellis Roger Williams Ohio State University/OARDC Ohio State University/OARDC

.. THE LEADERS

From top to bottom Dr. Thomas L. Payne Director, OARDC Keynote address Attentive audience to Dr. Payne's address Dr. Luther \\aters Chair, Horticulture and Crop Science

• il ~ THE BANQUET ~ il

• OWPA - TRADE SHOW AAAAAAAAAAAAAAAAAAAAAAAAAAAA

)

-OHIO WlNE PRODUCERS A.SSOCI :\TION

•

• THE CLASSROOM

Ellen Harkness Purdue University Microbiology workshop

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