Sciences ULS~ UNIVERSITY OF CALIFORN STACKS SJWAPES W. B. HEWITT CALIFORNIA AGRICULTURAL EXPERIMENT STATION BULLETIN 868 Studies on different rots of grape in California vineyards have been in progress over the past decade. This bulletin presents results of these studies and describes the kinds of rot found. The more common fungi associated with the rots and those known to be involved in rots are listed, and information on the ability of species of many of these fungi to infect grapes and cause rot is presented. Results of studies on the occurrence, sequence, and relative populations of spores of some of the most dominant grape-rotting fungi found on grapes in the vineyard are also presented. Special attention has been given to: • Summer bunch rot, a sour rot of grape clusters resulting from infection of grapes during bloom time. • Cultural practices influencing development of summer bunch rot. • Factors contributing to other rots of grapes. Early Botrytis rot of grapes also due to bloom time infection by airborne wind spores is described, and factors influencing the development of slip-skin and gray-mold diseases caused by B. cinerea are discussed. Results of studies on the development of Botrytis rots in California vineyards are given. The bulletin does not outline chemical control of rots but it does discuss cul- tural and other practices that should aid in prevention of rots. NOVEMBER, 1974 THE AUTHOR: William B. Hewitt is Professor of Plant Pathology Emeritus, Davis, and the San Joaquin Valley Agricultural Research and Extension Center, Parlier. [2] ROTS AND BUNCH ROTS OF GRAPES 1 INTRODUCTION Every growing season rots of grapes cause losses to growers, shippers, han- dlers, retailers, and consumers (Oga- wa et al. 1965). Rots develop in vine- yards, storage, transit, market and in the home of the consumer (Cicarrone et al, 1970; Harvey and Pentzer, 1960). The total loss due to such rots in any season is substantial, but is usually dif- ficult to evaluate. In individual vine- yards and in some districts, however, losses have ranged from a trace in some seasons to most of the crop in other seasons. Counts in different vineyards have demonstrated that losses caused by Botrytis rot ranged from a trace to over 75 per cent in the same season. Losses from summer bunch rot of Thompson Seedless grapes in individ- ual vineyards in the San Joaquin Val- ley have ranged from 5 per cent to over 36 per cent during the period 1958-1965. Rotting grapes may also affect the quality of wine if mixed in substantial quantities with sound grapes. Certain fungus rots, such as those caused by the fungi Rhizopus arrhizus Fisher, Aspergillus niger van Tiegh., and A. flavus Link., can have deleterious effects on musts or wine, and wine made from grapes rotted by combina- tions of fungi, yeast, and Acetobacter roseus Vaughn is also objectionable. Rotting grapes in vineyards often contain more than one microorganism and also include yeasts and Acetobac- ter. In California, in early season when grapes begin to ripen, those rotted by Botrytis cinerea Pers. ex Fr. (botry- tised) are seldom contaminated by other microorganisms. However, there are some exceptions; French Colom- bard grapes having early Botrytis rot, for example, may become infected with other organisms, especially if in- fested with yeast and fruit fly larvae. In late harvest season when rains have persisted for several days it is not un- common to find other organisms also associated with botrytised grapes. Pos- sible explanations for occurrence of rot caused by B. cinerea alone and not contaminated with other forms of rots are (1) the fungus is exceptionally aggressive and invades tissue rapidly and, (2) the fungus produces anti- biotic substances that may act as deterrents to other microorganisms (Ribereau-Gayon et al, 1952). When grapes which have been rotted by B. cinerea also become contaminated with other fungi, it is possible that the antibiotic substances produced by B. cinerea may have been diluted by grape juice or may have been washed out by rain. Most grapes with rot are a total loss, but in some European countries it has long been a practice to make special wines from botrytised grapes (Nelson and Amerine, 1956). Wines made from grapes partially decayed by B. cinerea have a distinct flavor and aroma and can command high prices. Some special wines are also made from botrytised grapes in California (Nelson and Amerine, 1957). Rots of grapes are often named after the fungus causing them. For l Submitted for publication February 5, 1974. [3] example, Botrytis rot is caused by B. cinerea, Cladosporium spot is caused by Cladosporium herbarum (Pers.) LK. ex Fr., and Phomopsis rot is caused by Phomopsis viticola Sacc, etc. Rots are also known by more descriptive terms such as slip-skin, smut, gray-mold, sooty mold, green mold, sour rot, etc. Some fungi re- ported to be capable of infecting sound mature grapes and causing rot are B. cinerea (Nelson 1951, a,b, 1956), Aspergillus niger van Tiegh. and Al- ternaria geophila Deszew (El-Helaly et al. } 1965). Glomerella cingulata (Stonem) Spauld. and Schrink is con- sidered to be one of the most impor- tant causes of rot (Lee, 1962). During harvest time in 1958 and periodically through 1958-1968 Cali- fornia vineyards were examined for rots of grapes, and grapes being de- livered to wineries were also examined for rots. This practice has continued and is becoming more common. At inspection stations in Napa Valley percentages of specific forms of rot were determined (Martini, 1966). Bot- rytis rot was the most prevalent of the rots found in the Napa and other north-coast county vineyards, but some degree of the disease was observed in most vineyard areas and in many loads of grapes being delivered to wineries. In the San Joaquin Valley, summer bunch rot has been a dominant form of rot found in clusters of Thompson Seedless grapes. In some seasons, wine grapes grown in the San Joaquin Val- ley may have considerable Botrytis rot as well as sour rot. Other forms of rot common in California vineyards are caused by species of Alternaria, As- pergillus, Cladosporium, Penicillium, Rhizopus and, in vineyards of some areas, Phomopsis. Some rot diseases of grapes not known to occur in California are: black rot caused by Guignardia bid- wellii (Ell.) Viala and Ravaz; anthrac- nose caused by Elsinoe ampelina (d. By) Shear, Sphaceloma ampeliuum (d. By); bitter rot caused by Melonco- nium fuligineum (Schribner and Vi- ala); ripe rot caused by Glomerella cingulata (Ston.) Spauld. and Schrink, etc. These diseases develop in areas where it rains during the growing season. Fortunately, California vineyards are not plagued with the downy mil- dew fungus Plasmopara viticola (B. and C.) Berl. and DsT., a serious foli- age pathogen which also causes disease of fruits and shoots. Downy mildew could probably become established in California given the proper set of conditions, i.e., environment, variety, locality, etc., and could do much dam- age in favorable seasons. Though a wild strain of the downy mildew fun- gus occurs in California on the wild grape Vitis California Benth. (Santilli, 1957), the strains affecting European grape varieties V. vinifera L. do not now occur in California vineyards. METHODS Standard techniques for culturing of microorganisms in tissues of grape and grapevine were used in this study. Culture media used were water agar (WA) and potato dextrose agar (PDA) made in the usual way but with the latter having only 10 grams of dex- trose per liter. Tissues were surface disinfected by submerging in solutions of either 65 per cent ethyl alcohol or sodium hypochlorite solution com- posed of 1 part of a proprietary so- [4] Fig. 1. A. Spores of Cladosporium herbarum, scanning electron micrograph at X5.500, showing marginal spines (arrow) observed on conidia in phase-contrast under the light microscope. Spores at extreme right of A were photographed at X500. B. Spores of Botrytis cinerea photographed at X500 under phase-contrast, light microscope. C. Spores of Alternaria tenuis; top row was photographed under phase-contrast at X500; lower groups were photographed under the light microscope at X60. D. Spores of Aspergillus niger. Left: scanning electron micrograph X5.500. Right: photographed under phase-con- trast X500. Arrow points to spines on spore margins corresponding to spikes shown on the two enlarged spores. E. Spores of Diplodia natalensis under light microscope at X60. dium hypochlorite compound contain- ing 5.25 per cent active ingredients plus 8 parts of distilled water plus 1 part of 95 per cent ethyl alcohol. Sub- mersion time in disinfesting solutions was from 3 to 5 minutes unless other- wise stated. Tissue pieces were plated directly after surface disinfesting di- rectly from the solutions onto media without rinsing in water. Flowers, grapes, and stem tissues were often disinfected and plated onto culture media in the vineyard. When tissues were taken to the laboratory for cul- turing they were placed in a cold chamber over ice soon after collecting and cultured within 12 hours. Fungus spores used for all studies were pro- duced in the laboratory on autoclaved grapes or on PDA. Fungus spores were trapped in vine- yards on glass slides, or on l/^-inch plastic strips 3-mounted on 3^-inch- diameter glass rods, or with Hirst 7- day spore traps whose exposed sur- faces were coated with a thin film of petroleum jelly. Plastic strips from the spore traps were examined with a microscope, and spores of certain spe- cies of fungi were identified on the [5] traps on the basis of morphology and ornamentation. The spores of C. her- baram as well as those of Alternaria spp. were positively identified by means of a light microscope (fig. 1). In some experiments clusters of flow- ers at pre-bloom and during bloom, and clusters of grapes at different stages of development, were inserted in brown paper bags (grocery type, 16 or 20 pounds capacity) to exclude in- sects and minimize contamination by fungi spores. Some of the bags were provided with 2- x 2-inch windows of clear polyethylene plastic sheeting for observing and to aid in placing inocu- lation at desired points (fig. 2). The sheeting, which was approximately 0.5 mils thick, was cut into 3-inch squares and mounted with masking tape over a 2- x 2-inch square hole cut in the side of the bag. Windows mounted this way lasted the season without tearing or loosening. On the vines, a strip of absorbent cotton \/ A - inch thick, 1-inch wide and 3-inches long was wound around the cluster stem at the union with the cane. The clusters were dipped in 65 per cent ethyl alcohol for slightly over 30 sec- onds and then inserted into bags while dripping wet. The top of each bag was gathered about the cotton pad- ding and securely tied with paper- coated wire. The tie was then ex- tended over the cane and again tied to Fig. 2. Thompson Seedless grapes en- closed in paper bags fitted with polyethy- lene film windows. Bags were used as in- oculation chambers and to exclude fungi and insects. keep the bag from slipping. Most of the paper bags retained their shape and seal for more than 120 days. FUNGI ASSOCIATED WITH GRAPES Preliminary work indicated that most grape rots were caused by fungi or that fungi were associated with rots as secondary organisms. To study them, fungi occurring naturally on grapes were determined by culturing flowers before bloom and during bloom, and from grapes at different stages of de- velopment. Grape flowers or young grapes, or both, were picked at ran- dom from clusters and cultured on WA for microorganisms as described in "METHODS," page 4. Some 70 species in 30 genera of fungi, as well as some bacteria includ- ing acetic-acid-forming bacteria, were [6] 100- Q £ 8 0- => b • ° 60- (/) w a. < 01 40- U_ O S* 20- / A — ^x^ 5 *— : Aspergillus -Alternaria Rhizopus- /* <-A. Penicillium **=«£ 4/7 5/l4 5/22 5/29 DATES SAMPLED mo/day ^8 Clodosporium 6/21 7/i 7/11 7/25 i7i Prebloom Full Shatter Small bloom pea STAGE OF DEVELOPMENT Grown 6% SS 10% SS 14% SS 18% SS Fig. 3. Per cent of Thompson Seedless grapes cultured showing presence of fungi at various stages of grape growth. Each point on the figure represents per cent of isolations out of 800 grapes tissue plated. cultured from grapes in vineyards of Napa, Sonoma, San Joaquin, Stanis- laus, Fresno, Tulare and Kern coun- ties, and in the Coachella Valley. Though the study has been in prog- ress since 1958 and a great many grapes have been cultured, most fungi recovered were isolated from grapes during the first two seasons. Since then other fungi have been isolated only occasionally. The following mi- croorganisms were most commonly isolated from grapes. (Not all fungi recovered were identified as to species, nor are they all listed.) Acetobacter roseus Vaughn and ap- parently other species; Ascochyta sp., * Alternaria tenuis Nees, Aspergillus niger v. Tiegh., black and brown spore forms, A. wentii Wehmer., A. flavus Link., A. ochraceus Wilhelm., Botrytis cinerea Pers. ex Fr., Candida sp., Chaetomium elatum Kze.,^ Clado- sporium herbarum Pers. ex Fr., Curv- ularia sp., ^^Diplodia natalensis P. Evans, Emericella rugulosa (Thorn. and Paper) Benjamin, Epicoccum sp., Fusarium moniliforme Sheldon, Fu- sidium sp., Helminthosporium sp., Heterosporium sp., Monilia sp., Ni- grospora sp., Popularia sp.,' Penicil- lium spp. (green)," Phomopsis viticola Sacc, Pullularia pullulans Berkh., Rhizopus arrihzus Fisher,^, stolonifer (Ehrenb. ex Fr.) Lind., Saccharomyces cerevisiac Meyen ex Hansen var el- lipsoideus, Stemphyllium botryosum Walker, Trichoderma lignorum (Tode) Harz., and Torula sp. A. tenuis, A. niger (the brown spored form), C. herbarum, R. arrhizus, R. stolonifer and a green-spored Penicil- lium sp. were the most dominant of the fungi occurring on grapes in the vineyards. Yeasts of various forms and Acetobacter sp. were prevalent in grapes with sour rots. Figure 3 shows occurrence of five dif- ferent fungi cultured from Thompson Seedless grapes at intervals through the 1972 season in vineyards of Fresno County. Populations of species of [7] Pre -bloom Full Shatter Small bloom pea size STAGE OF DEVELOPMENT Fig. 4. Per cent of Thompson Seedless grapes cultured from which Alternaria tenuis was cultured at different stages of development. Each point is the mean of 800 grape samples cultured from vines in each of three vineyards. Alternaria, Cladosporium and Asper- gillus rose rapidly at bloom time; As- pergillus and Rhizopus sp. continued to rise during the rest of the season whereas A Iternaria and Cladosporium, though erratic, tended to decline with the season. Figure 4 shows occurrence of A. tenuis on Thompson Seedless grapes from three different vineyards in Fresno County when cultured at intervals over the growing season. The fungus may have been present on the grapes in the form of spores on the surface, or as fungus mycelium in the grape tissue. This fungus increased rapidly just after bloom and then de- clined over the rest of the season. Apparently, the high numbers of this fungus in cultures following bloom were due to the growth and sporula- tion of fungus on flower anthers re- maining in the cluster after bloom. Tissue platings during the 1969-1970 seasons were also carried out in late fall, and showed that the spore load of Alternaria and Cladosporium, which had been relatively low, increased rapidly in October and early Novem- ber. Species of Rhizopus and Penicil- lium gradually increased during the season but their increase was much more gradual earlier in the season. In another experiment sticky tar- gets to catch fungus spores were set in five different vineyards, two planted in cultivars Thompson Seedless, and one each to Calmeria, Italia, and Rib- ier. The targets were placed in the area occupied by fruit and were lo- cated high, central, and low in each of three different grapevines in each of the five vineyards. Each target had three sticky surfaces (petroleum jelly on transparent tape) mounted on it; the sticky areas were always exposed in the same directions, generally east, northwest, and southeast. Traps were collected at intervals of 3 to 4 days. After collecting, the tapes were mounted on glass slides and the spores were counted by microscopic observa- tion. [8] 5/6 II 15 18 22 25 29 6/15 8 12 15 19 22 26 29 7^36 10 13 17 20 2427 31 8/3 7 10 14 17 21 24 28 31 9/3 DATE mo/day Fig. 5. Mean number of spores of Alternaria tenuis and Cladosporium herbarum col- lected and counted on 4.8 mm 2 of target area, (three stations, three directions each, at six locations in each of five vineyards). Targets first set on May 3, 1970, and reset on each of reading dates shown. Peaks in the linegraph (number of conidia) of A. tenuis for the single vineyard correlate with rain showers in this vineyard. Spores of Alternaria sp. were easily distinguished and identified by gross morphology. Culturing showed that A. tenuis was the dominant species of Alternaria present in the vineyards. Spores of C. herbarum were also dis- tinguished morphologically by micro- scope examination. These spores, (fig. 1), have characteristic shape and sur- face protuberances which permit rea- sonably accurate identification, as do spores of A. niger. Spore populations of A. tenuis and C. herbarum caught on targets were similar, so results were pooled and averaged for the five vineyards (fig. 5). The presence and amount of differ- ent fungi has been found to vary with vineyard and season, and in some vineyards to be influenced somewhat by adjoining crops. Cladosporium was more prevalent in a vineyard adjoin- ing an alfalfa planting and in those with grass cover, whereas greater amounts of Rhizopus and Penicillium fungi were found in a vineyard adjoin- ing a plum orchard; these last two fungi appeared to increase as plums ripened and began to drop. It was also observed that the incidence of Rhizopus was high in the vineyard adjoining sugar beets, and higher in that part of the vineyard next to the sugar beet field than in the part some 200 feet from the beets. Apparently, diversity of crops in the area may have considerable influence on the occur- rence of some fungi associated with rots. In some seasons many fungi were observed growing on old flower parts lodged in the clusters. In periods of high moisture these old flower parts were usually covered with spores of [9] fungi. Species of Alternaria, Clado- sporium, Stemphy Ilium, Aspergillus and Penicillium were most frequently recovered from them. Species of Botry- tis, Rhizopus and Trichoderma were also isolated frequently. INFECTION AND ROT INDEX To determine the ability of some fungi to infect grapes and to cause rot, sound, mature Thompson Seed- less grapes were positioned on paraffin- coated i/2-inch-mesh wire-gauze racks over water in moist chambers (fig. 6). Ten grapes were chosen at random from a lot set up in each of twelve treatments in each moist chamber. Table 1 shows treatments and results after inoculation with each of 17 spe- cies of fungi. Grapes in a single moist chamber were inoculated with spores of only one fungus, and tests were replicated three, four, and five times. Grapes were wounded by puncturing Fig. 6. Thompson Seedless grapes on a V^-inch-mesh hardware cloth screen folded at edges to fit in a plastic chamber; grapes were inoculated with Cladosporium herbarum spores. Treatments by row left to right, respectively, were: 1, 2, 3 = controls not inocu- lated, wet not injured, wet injured, dry not injured; 4, 5, 6, 7 = inoculated not injured, with grapejuice, in water 1,000 spores, 100 and 10 spores per grape; 8, 9, 10 injured- inoculated in water, 1,000, 100 and 10 spores per grape; 11, 12 inoculated dry, not in- jured. (This illustrates the method used to determine rot index and ability of fungi to rot grapes.) [10] 'A a en w Oh Uh L*j" and fungus used ( inoculum^) Per cent sugar Diplodia natalensis Asp( 'rgillus niger Rhizopus arrhizus A Uernaria tenuis Sound Injured Soun d Injured Sound Injured Sound Injured 5 2.4 3.4 1.6 0.5 6 2.3 2.8 2.2 0.6 7 1.9 2.6 1.8 0.3 8 1.8 1.7 2.3 0.2 9 0.3 1.7 1.9 0.2 10 0.1 2.9 1.5 1.8 0.1 11 — — 1.5 3.1 0.1 12 0.2 3.2 1.4 4.0 0.2 13 0.1 1.1 2.0 4.0 0.2 14 0.3 1.2 1.6 2.7 0.8 15 1.5 2.5 4.0 0.2 0.4 16 2.1 3.2 3.7 0.8 17 1.8 4.0 3.0 0.1 4.0 1.1 18 2.6 4.0 3.4 4.0 0.5 1.0 *Refractometer readings of total soluble solids of grape juice expressed as per cent sugar. "fRot index is mean score based on per cent of the surface of the grape that was infected and rotted: 0=no rot; 1=5%; 2=5-15%; 3=15-50%; 4=50%. 4^Drop of spore suspension in water placed on berry and incubated in moist chambers. Incubation period =10 days at 24° C. [13] tion of Eosin Y) ranged from 3 to as many as 16 per grape. The mean num- ber of cracks in 30 grapes was found to be 7.8. Though soaking grapes in water with Eosin Y stain demonstrated that water may induce development of small cracks in the skin of Thompson Seedless grapes, and that the number of cracks increased with time in wa- ter, there is some question as to whether the cracks influenced infec- tion and rot. Had the presence of cracks increased infection and subse- quent rot of grapes (table 1), then more of the fungi should have in- duced rot and more rot should have developed on grapes inoculated by means of spores suspended in water. A . tenuis, A. niger (brown conidial form) and R. arrhizus are commonly associated with rot in the vineyard. Furthermore, these fungi induced rot in some grapes when infested with conidia in a water suspension (table 1). An additional experiment was set up to determine if cracks had any significant influence on infection and rot. Sound, mature Thompson Seed- less grapes were chosen at random from a lot that averaged 22 per cent sugar; each test run contained 120 grapes. Grapes were placed on l/g-inch mesh wire hardware cloth in moist chambers. Treatments were (1) 0.5 ml of water, (2) 0.5 ml of Thompson Seedless grape juice, and (3) 0.5 ml of grape juice plus 1,000 conidia. Read- ings were made on the 5th day. Table 4 shows results as per cent of grapes affected, with cracks in treatments 1 and 2 and with rot in 3. Since myce- lium of R. arrhizus overran much of each grape in a 5-day period, and as it appeared that the fungus may have infected through the pedicel, the in- oculation part of the experiment with R. arrhizus was repeated. In the above experiment the per cent of grapes with cracks in the skin (as demonstrated with the Eosin Y test after 5 days of the experiment) was greater under water than under grape juice. The per cent of grapes with rot greatly exceeded the per cent with cracks. The results indicate that conidia of these fungi under such conditions do not necessarily infect the grape through cracks, but that some do infect by enzyme action and degrading of the cuticle or by other modes of penetration. In these tests, infection by A. tenuis appeared to be a result of enzymatic action because the cuticle of the grape under the in- oculation drop was etched. The results of these tests indicate that under ideal conditions juice drip- Table 4 PER CENT OF THOMPSON SEEDLESS GRAPES FORMING CRACKS IN SKIN UNDER A DROP OF WATER OR GRAPE JUICE. AND PER CENT OF ROT CAUSED BY AN INOCULATION OF CONIDIA IN GRAPE JUICE. C. mtrol | not inoculated (!) Per cent Inocu lated. 1000 spores each: Per cent Fungus of grapes with areas i cracks treated in skin under with: of gra pe ro t after inoculation in drops of grape juice Alternaria tenuis water 13.3 grape juice 10.0 65.8$ Aspergillus niger ( brown ) 18.3 11.3 53.3 Rhizopus arrhizus* 1 a) 6.7 0.0 40.0 Rhizopus arrhizus 1 l>) 15.0 - 49.0 *In the experiment |a| the fungus grew rapidly over the grape in this test and infection occurred through the grape pedicel: the stem ends of a second set of grapes were lb) inoculated but not tested for cracks. In (b) there were no infected pedicels. TEach fungus was tested in a separate moist chamber therefore a control was run in each chamber for each fungus. There were 120 grapes in each control and each inoculation. Readings were made on the 5th day. IpForty per cent of the infected grapes developed a typical brown ring, indicating enzyme degradation of grape cuticle. [14] ping from grapes in a cluster in the vineyard may serve as a medium for growth of fungi such as species of Aspergillus and Rhizopus, and that under such conditions these fungi can cause rot of grapes. It may be in this way that R. arrhizus becomes such an important factor in sour rots of clusters. It is evident that some of these fungi are therefore capable of direct infection through the uninjured skin and may be classed as primary rot or- ganisms. Other fungi that infect grape tissue only through wound or rot le- sions caused by other fungi are classed as secondary rot organisms. Some primary rot fungi found in California vineyards are A. tenuis, B. cinerea, C. herbarum, D. natalensis, and P. viticola. Under very favorable conditions, which would be in water or grape juice, A. niger and R. arrhi- zus may also be primary rot organisms. However, these last two fungi gener- ally are secondary rot fungi. Some of the other secondary rot organisms are A. flavus, other Aspergillus species, Curvularia sp., Penicillium sp., and R. stolonifer. SOUR ROTS OF GRAPES Sour rots of grapes are characterized by a pungent sour vinegar odor. There are generally a complex of microor- ganisms associated with these odors, and they may be involved in the de- velopment of sour rots. Organisms most commonly associated with sour rots are species of Alternaria, Asper- gillus, Caldosporium, Diplodia, Peni- cillium, Rhizopus, yeast, Acetobacter, and some other bacteria, fruit flies, and dried fruit beetles. The odor comes from conversion of sugars to alcohol and then to acetic acid. Sour rots may be grouped into two general forms on the basis of over-all symptoms; those which rot a large pro- portion of the center of the cluster (typically a bunch rot), and those which rot only a few grapes or cluster- ettes on the shoulder side or tips of clusters. Rots of the center of the clus- ter may again be divided into two groups for example: (1) summer bunch rot caused by D. natalensis sour rot sometimes follows bunch rot caused by B. cinerea (Ciccarone, 1970), and (2) sour rots caused by other micro- organisms which may enter grapes through wounds. Summer bunch rot: the disease Summer bunch rot in its late stages of development is composed of a com- plex (many different microorganisms) and is difficult to distinguish from other forms of sour bunch rot (Plate I:A,B,C-see pages 26, 27). It has been found mostly in Thompson Seedless and to some extent in grapes of other cultivars such as Perlette, Delight, Black Monukka, Red Malaga and oc- casionally in Emperor (Hewitt, 1962; Hewitt et al., 1962). Development of summer bunch rot disease is started by D. natalensis (Hewitt, 1962; Strobel and Hewitt, 1964). This fungus also causes a cane blight on the same vari- eties (Plate I:E,F), a canker disease originating from infection through pruning and girdling wounds (fig. 7), and heart rot and trunk-wood decay. The initial causal fungus was first identified as D. viticola Desm, because it fitted the general description and was isolated from grape (Hewitt, et al., 1962). After a more detailed study the fungus was identified as D. natalensis (Barb and Hewitt, 1965). [15] Summer bunch rot is characterized by a strong vinegar odor, excessive dripping of juice from rotting grapes in the center of the cluster, and the presence of numerous fruit flies, dried fruit beetles, and larvae. In the vine- yard one can distinguish summer bunch rot initiated by D. natalensis from other sour rots by association with Diplodia cane blight (Plate I:D,E,F). The two diseases occur to- gether. Diplodia cane blight and cank- er diseases are described on page 29. Infection by Diplodia occurs at bloom time. This fungus infects the grape ovary during bloom. Spores that light on the flower stigma germinate there and the germ tube grows down the blossom style and from there into the young grape. Tissue platings of infected blossoms and subsequently developing grapes indicate that the fungus enters the stigma and traverses the style (fig. 8) into the grape (Strobel and Hewitt, 1964). The styles dehisce (drop) from most of the grapes of Thompson Seedless during growth, leaving only a stylar scar. Infections which have progressed only partly along the style before latency are de- hisced with the style as the grape grows. Infections which have pro- gressed past the zone of abscission (de- hiscence) of the style into the grape before latency is initiated remain and later cause rot. At what time and place the fungus bridges the style into the ovary has not been determined. The fungus remains latent in the tis- sue at the base of the style until about the time grape sugars reach some 10 to 14 per cent, at about which time the fungus resumes growth and causes rot of infected grapes. Diseased and rotting grapes first turn pinkish, then the skin cracks, and juice leaks out. The fungus will grow out through the cracks in the skin and spread to adja- cent grapes. At this stage, summer H lifV-.V/^ •;; Fig. 7. Diplodia canker caused by D. na- talensis on trunk of Thompson Seedless grapevine. Infection occurred through the girdle injury (groove around trunk). bunch rot (caused by D. natalensis) may be determined with certainty on the basis of symptoms. The skin of rotting grapes will have a pink color and fungus seen in the cracks will be white. The fungus may also be iso- lated in pure culture at this stage-of rot. At about this stage of develop- ment (or even a little earlier, when the grapes crack), the fruit fly Droso- phila melanogaster Meig. and dried fruit beetles such as Carpophilus hemipterns (Linn.) start to visit the cracked and rotting grapes. In visiting cracks in grapes these insects also lay eggs in the cracks and leave spores of fungi, yeast, and bacteria. As the or- ganisms grow in the grape they cause it to form juice; the juice drips out of the diseased grapes and wets other grapes and in turn causes more grapes [16] WW i Fig. 8. Cross section of Thompson Seedless grape stigma and style. A. Shortly after bloom; note spore (arrow) of Diplodia natalensis at top on stigmatic surface. B. At about shatter time; note cell division occurring at arrow. C. Twenty days after bloom, showing necrotic style; arrow points to pieces of D. natalensis spores (note also the layer of cell- division forming between style and ovule). [17] to crack. Juice from the dripping grapes is also a good medium for the growth of many fungi which occur naturally as fungus spores on grapes and flower parts throughout the clus- ter. By this time the rot has become a complex of microorganisms and will smell of vinegar. After these organ- isms start rotting the grapes and vine- gar forms, the fungus D. natalensis will die out or become very difficult to isolate from grapes of rotting clus- ters, or both. The cause of summer bunch rot The first indication of the nature of the cause of summer bunch rot was determined in the summer of 1963. Several vineyards in which the disease was known to be severe were observed regularly, and tissues of the grapes were cultured at intervals to deter- mine early stages of the rot. At inter- vals from about 10 days before bloom until a few days before harvest, flow- ers and grapes were cultured on water agar in Petri plates. Flowers and later grapes were taken at random from the clusters and plated without surface disinfecting into water agar. Table 5 shows results of culturing flowers and grapes in three Thompson Seedless vineyards at intervals from a time pre- bloom until the grapes reached about 18 per cent sugar. Though many fungi were cultured from flowers and grapes of the three vineyards, the dominant fungi found were species of Alternaria, Aspergillus , Cladosporium, Diplodia, Penicillium and Rhizopus. The frequency of their occurrence varied with the vineyard and the collection period of the sea- son. The high incidence of D. natalen- sis in late season in vineyards D and L may have been due to the accumulated Table 5 RESULTS OF CULTURING FUNGI FROM FLOWERS AND GRAPES ON THOMPSON SEEDLESS, EXPRESSED AS PER CENT OF CULTURE PLATES WITH FUNGI CULTURED* Culture d prior to grape maturity Cultured when grapi sugar content was: Time of culture Fungi cultured Pre- Full Shatter Grapes Thinning half- ( genus) bloom bloom time time grown 6% 10% 14% 18% Viney ardD A. tenuis 39 64 24 35 65 7 20 — A. niger 1 45 44 23 90 97 98 — C. herbarum 19 66 38 — D. natalensis 2 1.5 12 9 5 — Penicillium sp. 8 23 12 11 3 9 2 3 — R. arrhizus 1 8 6 6 3 19 65 32 - Vine' yard L A. tenuis 31 70 76 67 53 — 66 3 3 A. niger 30 40 45 84 — 42 86 97 C. herbarum 4 8 20 5 — 8 D. natalensis — 2 11 3 Penicillium sp. 18 20 6 4 5 — 50 46 75 R. arrhizus 10 18 4 12 16 - 14 27 61 Vine) ard M A tenuis 38 86 94 33 96 — 22 52 — A. niger 4 32 63 61 92 — 96 90 — C. herbarum 20 72 86 6 84 — 74 — D. natalensis 2 2 4 — 2 4 — Penicillium sp. 14 6 10 — 2 2 — R. arrhizus 2 18 8 14 - 43 72 — *Cultures were made in flowers and grapes in three vineyards: D near Arvin. Kern County: L 4 mi. northeast of Selma; M 3 mi. north of Caruthers in Fresno County. [18] spore load on grapes. Spores of D. natalensis are dispersed with dust, and repeated cultivation after bloom cre- ates such dust. Organisms isolated most frequently from clusters of rotting grapes were species of Aspergillus, Rhizopus, sev- eral yeasts, and bacteria. Larvae of fruit flies or dried fruit beetles were usually present. D. natalensis was iso- lated in tissue platings of flowers and grapes at intervals from full bloom until pre-harvest time. The fungus was not cultured from grape tissues pre-bloom, and its occurrence in cul- tures varied with the vineyard and the time of season at which the grape tis- sues were cultured. To determine which organisms or combinations of organisms most com- monly associated with the rot could induce rot of grapes or cause the con- dition or complex known as summer bunch rot, exploratory tests were first set up in the laboratory. Clusters of Thompson Seedless grapes were dipped in 65 per cent ethyl alcohol to decontaminate their surfaces, then they were hung on racks and covered with polyethylene bags in the labora- tory. Table 6 shows treatments and results. Some treatments were repli- cated two, three and four times. In- juries were made by piercing four or five grapes in the upper shoulder of the cluster with a laboratory dissect- ing needle inserted through a side of the polyethylene bag. Inoculations with spore suspensions were made with a hypodermic syringe or the nee- dle, or both. D. melanogaster fruit flies free of organisms were reared in the usual manner in the laboratorv. These laboratory studies showed that under humid conditions in poly- ethylene bags, A. niger caused rot of injured grapes. D. natalensis and R. arrhizus caused rot of both sound and injured grapes. Fruit flies alone with- out yeast but in combination with A. niger, D. natalensis, or R. arrhizus did not develop on grapes. Fruit flies placed in cages with grapes that had been injured and inoculated with yeast increased in numbers; the larvae use yeast as food and do not develop without it. Combinations of D. natal- ensis, yeast, fruit flies, and R. arrhizus and yeast caused a rot of clusters much like that observed in vineyards. Upon examination of grapes in the control cluster (and also in clusters in other treatments) it was found that some grapes had cracked and that some of these had become infected with A. niger and R. arrhizus. Further- more, it was difficult in the laboratory to contain these latter two organisms. Table 6 shows that A. niger and R. arrhizus occurred frequently in com- binations with some other organisms, even though attempts were made to exclude them. The degree of rot, how- ever, caused by A. niger and R. arrhi- zus was small. Results based on num- bers involved indicated that D. natal- ensis was most likely to initiate rot of grapes in the cluster. Further studies were carried out in a vineyard in Kern County. There experiments were to determine the organisms that initiate summer bunch rot. Brown paper bags (as described in "METHODS" and shown in fig- ure 2), were used to cover clusters and exclude various microorganisms. Inoculations with fungus spores or yeast were made by inserting a hypo- dermic syringe needle through the side of the bag. Fungus spores, either dry or wet, were discharged into the bag by means of the syringe. Injuries to grapes were made by puncturing four or five grapes in the top shoulder of a cluster either with a syringe nee- dle or the needle of a laboratory dis- secting instrument. Fruit flies were inserted through cuts in the side of [19] Q W W > O O w oo Oh CD S 52 wz Ot/)Q § u CD^ °^ z< o^ ^s Ohcd Cl, cj; x< Wc_> III Eo S » + + I tttt I + + I I '+ + + + I + + + + + • I I I I I I I I I I m m m m m m o m m m m m m 2 o o o o n Tf m h io po o •— i o Cvl o cm ■* r-n en cm ^ —i m ■s I I I IIIIS i in in oj q "2 © d rA cm I I I II I I II I I I I I I I I I I I I I I I I I I I I ■* i-H CM H coTfcsi^vO'*co^-^tco Tt ^ * ■* * * e e 8 ^ =», « a , Q Q Q£ Q Q DC >- > Q ^11 X = call £-g u 3 00 - 3 _2 c u'^ £ u c/)^ £«ll . S ocm S II 1 + ra jj in -J Si!" is jj O BC^H 2-9-e ; c cq+ [20] C f. w C go -I M^ Oh cfiO S< g* en cr; en >r dQ u H C ■5 o II 5 : in o co o Ln CM CM CO — i CO 00 CM CO 00 00 ^fj LO CO t- CO c> io C? ^ t~ -h -f o e o o o'oooof-cooooo [21] the bag. After use, all entry punctures of the paper bags were sealed with masking tape. Clusters of Thompson Seedless grapes were bagged 5 days after hand-thinning the clusters, a normal operation in preparing and growing fresh table grapes (Winkler, 1962). Inoculations were made 14 days before harvest when grape sugars were near 15 per cent. Table 7 lists treatments and shows results of readings taken at harvest time. Diplodia rot developed in clus- ters of grapes that were not injured and inoculated with spore suspension of D. natalensis alone, in grapes not injured, in clusters in which grapes were injured, in clusters inoculated with combinations of D. natalensis, fruit flies, and yeast, and also in clus- ters inoculated with A. niger and R. arrhizus. D. natalensis also caused rot in clusters of the control series, in clusters that were injured and not inoculated, and in clusters inoculated only with spores of A. tenuis and R. arrhizus. The only time that these clusters in paper bags were exposed to infestation by D. natalensis was be- fore the clusters were dipped in ethyl alcohol and bagged. Rot development in this test indicated that infection of grapes with D. natalensis took place some time before the clusters were placed in paper bags. Results of inoculations showed that A. niger caused rot of only a few grapes in the clusters, and that there was more Aspergillus rot in clusters of injured grapes than in clusters of grapes not injured./ R. arrhizus often spread and rotted much of the cluster, and caused more rot of grapes in clus- ters with injured grapes than it did in clusters of uninjured grapes, f Fruit flies developed in bagged clus- ters only when injured and yeast was present. Clusters inoculated with D. natalensis, fruit flies, and yeast devel- oped internal rot and decay; some grapes in these clusters had typical Diplodia rot, but most of them were destroyed by fruit flies and yeast. Clusters in which grapes were injured and then inoculated with R. arrhizus, fruit flies, and yeast were virtually de- stroyed by a complex rot which re- sembled summer bunch rot in many respects. Clusters in which grapes were not injured before inoculation with D. natalensis, A. niger, and R. arrhizus also developed a bunch rot similar to summer bunch rot (table 7). Although some of the paper bags became contaminated with other fungi and with fruit flies or dried fruit bee- tles, or both, there were few of these contaminations. However, in the last treatment listed in table 7 (infection was with D. natalensis, A. niger, and R. arrhizus), contamination with fruit flies and dried fruit beetles was high, perhaps due to decay activity in the bags which attracted fruit flies and dried fruit beetles. It was concluded from this study that the summer bunch rot complex was most likely initiated by D. natal- ensis (Barb and Hewitt, 1965) and this fungus started rot of some grapes in the clusters and that other organisms followed. It was also demonstrated that if grapes in a cluster were in- jured, then Rhizopus could initiate the development of summer bunch rot. Infection period Culturing of tissues of flowers and grapes at intervals during the growing season (table 5) showed that in some vineyards D. natalensis occurred on grapes early in the season shortly after bloom time. Results of the experi- ment (Plate I) in which clusters of Thompson Seedless were placed in brown paper bags just after shatter time, and inoculated 14 days before [22] harvest show that 22 of the 67 control clusters had Diplodia rot. While in bags these clusters were protected from contamination by spores of fungi. Infection by D. natalensis prob- ably took place before surface disin- fection and bagging. Time of infection To establish time of infection by this fungus, clusters of Thompson Seedless grapes were disinfected by dipping them in 65 per cent ethyl alcohol and bagging them as previously described at intervals from pre-bloom until grape sugar was about 14 per cent. Other sets of clusters were bagged pre- bloom and later inoculated with spores of D. natalensis at full bloom, shatter time, and thinning time. Inoculations were made by dusting spores of D. natalensis into the cluster through an entry hole in the side of the bag. The holes were sealed with masking tape after inoculation. Table 8 shows combined results of bagging tests in three different vine- yards. Clusters bagged at pre-bloom had no rot, whereas clusters bagged during full bloom and at subsequent times developed Diplodia rot and rot complex. (Some other rots also devel- oped.) Clusters which had a rot com- plex were usually infested with fruit flies or dried fruit beetles, or both. These and other contaminants appar- ently gained entry into some of the bags before harvest time. The experiment indicated that in- fection by D. natalensis occurred during bloom time. Decline in the incidence of Diplodia rot in clusters bagged after thinning time is believed to be due either to shelling of infected grapes with shatter, or to drying and abscission of infected styles as grapes matured, or to increase in rot complex due to contamination of bags with flies and beetles. It was noted in this exper- QE >* H CCCC w P Jh DO < c/) H JZ Q< W W c/2 O O CO CO |21 o m co co x 2<~ > o o H o J J iO M O O O ! g Z £ c -g g£ H c CT3 .s .. 77 38 200 100 109 54 2 190 95 105 52 158 79 7 3 3 189 94 117 58 172 86 43 21 • 4 182 91 115 57 190 95 83 41 7 62 31 90 45 119 59 50 25 11 70 35 132 66 46 23 72 36 17 22 14 2 1 21 13 2 1 25 2 1 2 1 1 0.5 1 0.5 *Flowers were inoculated at full bloom with spores of B. cinerea either suspended in water or dry. Samples for plating were collected at random from several clusters on intervals indicated after inoculation. Some flowers and young grapes were plated directly on water agar ( natural) and others were surface-djsinfected before plating and culturing. fTwo hundred flowers or young grapes were plated on each collection date except for the 17th day when only 160 were plated. undisinfected flowers and grapes, whereas the fungus could have grown only from infections in the disinfected flowers and young grapes. The inci- dence of infection increased after the second day to about 50 per cent of the flowers and remained constant through the 11th day; it then decreased mark- edly, and on the 25th day only about i/ 2 to 1 per cent of the remaining grapes were infected. In the San Joaquin Valley, styles of Thompson Seedless grape flowers nor- mally appear to dry and to drop from grapes during their rapid-growth phase. Observations of B. cinerea growth from Thompson Seedless grape tissue in the culture indicates that the fungus was confined to the stigma and style. Apparently, fungus growth was arrested, probably by defense mecha- nisms in tissues as it progresses in rot of the style and before entering the grape. Figure 8 shows the stigma and style of a young grape a few days after bloom; later a cork layer forms next to the grape and the style becomes separated from the grape. The area where the style separates from the grape (abscission zone) is lighter than the tissue of the style and grape. In many infections the fungus is arrested and becomes latent in the style, and when the style drops during the rapid- growth phase of the grape the fungus drops with it. However, in experimen- tal inoculations of flowers of Thomp- son Seedless a few fungus infections advanced to the point in the stylar end of the grape where they were not shed with the style. The fungus in these latter infections broke latency (i.e., overcame the defense mechanism of the green grape) and caused rot as the grape matured. Cluster rot can be caused by one grape only because the fungus will grow rapidly from one grape to others. In experimental work carried out in Napa Valley in 1971 and 1972 on time of infection and latency of B. cinerea (McClellan, 1972 and McClellan et al., 1973), reported that in 1972 more than 90 per cent of the styles of flowers on Pinot St. George and 50 per cent on Sauvignon vert were infected with B. cinerea. The styles of flowers of grapes in the Napa Valley did not drop as they did on Thompson Seedless in the San Joaquin Valley. By harvest time in 1972 B. cinerea was still viable in most of the old dried styles still at- tached to the grapes, although only 2 per cent of the clusters of Pinot St. George and 9 per cent of Sauvignon vert clusters developed early Botrytis [41] rot. In 1971 in the same two vineyards Pinot St. George had 60 per cent clus- ter rot and Sauvignon vert had 25 per cent. McClellan and Hewitt (1973) showed that B. cinerea entered the grape flower through the stigma and then moved into the style. They also showed that the fungus usually causes a decay of the style; the fungus becomes latent in the style but remains viable through most of the growing season. In these two varieties the fungus moved from the style into only a small percentage of the grapes, and there to cause rot. Neither the path of the fungus during infection nor the time of infection of the berry was determined. In other work on the cultures of flowers and young grapes of Grey Riesling, Pinot St. George, Sauvignon Blanc, Sauvignon vert and White Riesling made from 1966 through 1969 it was noted that B. cinerea al- ways grew out of the stylar end (fig. 14). In culture, Botrytis rot of the berry whether very young or in any stage of development to nearly full grown always started at the stylar end. The fungus was also cultured from flower parts stuck to the sides of grapes. These observations were con- firmed by McClellan and Hewitt (1973). Only a small percentage of the grapes in cultures and in the vineyards developed Botrytis rot. The develop- ment of rot of grapes in culture col- lected at various stages of development suggests that the fungus crossed from the style to the grape early in fruit development. Sugars and amino acids from the surfaces of mature grapes improve the germination of Botrytis spores and growth of the germ tube. McClellan and Hewitt (1973) showed that ex- tracts of the grape flower style also greatly improve spore germination Fig. 14. Botrytis cinerea on grapes. Left: fungus growth and sporulation on style of young grape. Right: sporulation of B. cinerea on surface of a diseased young grape. Note how fungus forms spores on long stalks (conidiophores) high into the air where they can be blown away by wind. [42] and germ-tube growth, and grape stigma and style tissues are good me- dia for development of the fungus and consequent infection of the grape. Application of extracts of the young grape taken at shatter time greatly re- duced germination of spores and markedly depressed growth on the spore germ-tube (McClellan and Hew- itt, 1973). These effects continued until grapes were some 10 mm in diameter. Extracts made from older grapes had little effect. It is probable that young grapes have a built-in mechanism for defense, and that the defense system serves to induce latency in the fungus. Experiments on control of early Botrytis rot have provided additional evidence indicating that the fungus infects the grape at bloom time. When benomyl ([methyl-1- (butyl carbamoyl)- 2-benzimidazole carbamate]), is ap- plied when flowers first start to open, it has often given 90 to 95 per cent control of this rot; when applied ear- lier or later the chemical was less effective. Benomyl is a systemic fungi- cide (it is absorbed into the plant and then translocated) and thus moves into the style and stylar end of the grape flower ovary. Applied at early bloom stage, it accumulates at toxic levels; applied earlier or later, con- centration levels are lower and control is less effective (McClellan, 1972). Apparently, grapes inside naturally- infected clusters in the vineyard are more prone to rot than are grapes on the exterior of the cluster; although there are as many infected grapes on the outside of a cluster as there are on the inside, grapes on the inside are perhaps in a slightly more favorable micro-climate for the fungus than are those on the outside and therefore more prone to rot. An indication of what higher humidity may do is illus- trated by grape clusters confined in paper bags from post-bloom or past- shatter; the bagged clusters developed 14 per cent more Botrytis rot than did unbagged ones. In the course of this study (1959 through 1971) flowers and grapes at varying stages of development were cultured for microorganisms of all sorts. Grape varieties included: Cari- gnane, Chenin blanc, Emperor, Fran- ken Riesling, Grey Riesling, Grenache, Petite Sirah, Pinot Noir, Pinot St. George, Sauvignon Blanc, Sauvignon vert, Thompson Seedless, Tokay, White Riesling, and Zinfandel. Tissue platings of flowers and grapes show that all the varieties become infected with B. cinerea during bloom time, and that the fungus becomes latent in the style or stylar end of the grape and remains dormant there until the grape reaches a stage of maturity permitting the fungus to resume growth and start rot. The stage of maturity at which rot will begin varies with the grape vari- ety, the locality, and the season. For example, Grenache grapes will rot in late June, July, and early August, Thompson Seedless grapes will rot about or during harvest time, and Emperor grapes will usually rot dur- ing harvest or in storage or transit. Grapes of many of the wine varieties grown in Napa, Sonoma, and Santa Clara counties usually developed early Botrytis rot later in the season than did the same varieties grown in the San Joaquin Valley. Incidence of early Botrytis rot The amount of early Botrytis rot in a grape variety also will vary with the vineyard, the locality, and the season. During 1963 in an experimental plot of Chenin blanc, 25 per cent of the grape flowers were infected with B. cinerea at shatter time whereas 9.7 per cent of the clusters developed rot before harvest time although there were no rains during late season. Zin- [43] fandel in a vineyard near Lodi had 71 per cent of their grape ovaries in- fected at shatter time and only 1 per cent of the clusters had rot before harvest time. In 1967 in a Grenache vineyard in Stanislaus County, 92 per cent of the grapes were infected with B. cinerea when they were about 14 - inch in diameter, and later in July 33.4 per cent of the clusters had de- veloped rot. In Napa Valley in 1959 the per cent of clusters of grapes with early Botrytis rot in Sauvignon vert ranged from 0.2 to 4 per cent, in White Riesling from 5.9 to 7.7 per cent, in Petite Sirah from a trace to 20.4 per cent, and in Zinfandel from a trace to 17.4 per cent. In 1963 in the same area, vineyards of Sauvignon vert rot incidence ranged from 1.5 to 20.1 per cent. Sour Botrytis rot In some years, especially after a sum- mer rain or during times of high hu- midity, clusters of grapes with early Botrytis rot may also develop sour rot. Such clusters become infested with fruit flies, dried fruit beetles, yeast, bacteria, and other fungi, and the rot may destroy most of the Botrytis in- fection. When there is no rain before harvest, grapes with early Botrytis rot usually dry up and mummify. Slip-skin Slip-skin disease is an early stage in the development of Botrytis rot (Nel- son, 1956). At this stage small cracks in the skin are usually evident, and when the grape is rubbed slightly its skin breaks and slips away from the pulp. Later, the fungus grows out through the cracks in the skin and produces gray tuffs of spores on branched stalks. When grapes are ready for harvest, slip-skin disease will develop on grapes 3 or 4 days after a rain. Although the first fall rains (especially if brief) sel- dom result in much Botrytis rot, they do stimulate fungus sporulation and result in distribution of spores through- out portions of the vineyard. It is dur- ing subsequent rains that infection us- ually becomes extensive. Botrytis rot can be expected to increase with dura- tion of rain and with repeated rains. Infection of grapes by Botrytis Experiments show that under natural conditions B. cinerea will penetrate directly through the grape cuticle (wax coating) and infects its skin cells. The spores germinate by extruding a short germ tube which turns up and then, after a short growth, turns down to form a hook so that the end of the germ tube faces the grape skin at right angles to its surface. The end of the germ tube then presses against the surface of the grape and enlarges into an appressorium (suction cup). Then, directly in the center of the appres- sorium, a small peg-like structure (in- fection peg) is formed (fig. 15) and forces its way through the grape cuti- cle (the process appears to be a me- chanical one). Just below the cuticle on the surface of the epidermal cells the infection peg flattens to form a subcuticular vesicle (an enlargement) (Nelson, 1956). As this vesicle forms, the entire contents of the conidium flow through the germ tube down the infection peg and into the vesicle; after the vesicle fills, a wall is formed in the fungus at the union of peg with the vesicle. At about this stage of de- velopment the vesicle forms a side growth that gradually becomes a branch of the fungus (hyphum); the hyphum then grows from the vesicle and branches and spreads intercullu- larly in a normal way among the grape skin cells. The cytoplasm (contents of the hyphae) of the fungus extrudes enzymes over the epidermal cell of [44] Fig. 15. Mode of direct infection through cuticle and outer skin of Tokay grape. Left: section through a conidium (C), cuticle (ct), and epidermal cells of grape. The conidium germ tube (t) has formed an appressorium on the cuticle and forced an infection peg (at tip of arrow) through the cuticle. Right: infection peg is shown at tip of upper arrow; below this on the epidermis a subcuticular vesicle (SV) has formed and extended between epidermal cells. (Photo courtesy of Klayton Nelson.) the grape. These enzymes degrade the intercellular pectic materials that ce- ment the grape tissue cell walls to- gether. The fungus permeates the skin tissues, which are approximately eight cells thick, and spreads between the skin and pulp before entering and de- grading the pulp (Nelson, 1956). In- fected skin tissues will crack at the surface and permit moisture to evapo- rate, and as these areas enlarge the fungus grows out through the cracks and sporulates. In the early stages of infection changes in skin color can be seen on the surface of the grape. After invad- ing much of the skin tissue the fungus invades the pulp of the grape. In the grape tissue and on the surface, the fungus forms heavy, compact, irregu- lar mats of hyphae with specialized surface cells that become black; these are called "sclerotia" and they may form on cluster-stem tissues as well as on grapes. Moisture and temperature effects. Spores of B. cinerea will germinate in water, but do so more readily in fruit juice (Kosuge and Hewitt, 1964; McClellan and Hewitt, 1973; Nelson, 1951 a,b). Spores dusted on grapes, or wetted and then sprayed on grapes which were afterwards dried, were found to germinate and infect grapes at relative humidities of about 80 per cent and higher (Nelson, 1951 b). Ger- mination and infection was found to increase at higher humidities, and longer wet periods at relative humidi- ties below 95 per cent before drying the grapes also increase germination and infection. The spores germinate and infest most readily when relative hu- midity is about 94 per cent or higher (Nelson, 1951 b). Spores will germinate and infect grapes at temperatures which range from 3 to about 30° C. Infection of Tokay grapes will occur within about 12 to 24 hours at 12 to [45] 20° C when spores are seeded onto grapes in water. Infection periods be- come longer as temperatures decrease below 12° C; for example, at 3° C infection of Emperor grapes takes from 60 to 72 hours and about 30 hours at 9° C (Nelson, 1951 b). At higher temperatures the germination rate of spores slows and infection de- creases. The fungus will infect directly through the cuticle of the grape (Nel- son, 1951 b). Injuries or small cracks following seeding with spores in the skin do not appear to influence the amount of infection and rot. Grapes with high sugar content are more prone to infection and development of slip- skin and Botrytis rot than are grapes with low sugar and high acid content. Gray mold. This name is given to Botrytis rot when it develops during storage at low temperatures. The fun- gus grows over the surface of grapes and from grape to grape in the clus- ter, forming a mat of mycelium having a gray cast. Carry-over of Botrytis cinerea B. cinerea is an ever-present organism in all vineyard districts, but evidence suggests that the fungus is more abun- dant in some vineyards than in others. Perhaps this is related to climate, cul- tural practices, or combinations of these factors. The fungus has been observed to over-winter and over-summer in the form of black sclerotia on old cluster stems, on canes, and on mummified grapes. The sclerotial structures serve Fig. 16. French Colombard grape cluster remnants mummified by B. cinerea and picked from grapevines in the spring. They have mummified grapes, sclerotia, and spores and serve to build-up inoculum in the vineyard. to carry the fungus through unfavor- able periods of summer and winter (fig. 16). It is believed that sclerotia of this nature are one of the principal sources of the spores that infect dur- ing bloom time; in wet periods of moderate temperatures these sclerotia will produce large numbers of spores, which can be wind-borne and blown about in the vineyards. Because B. cinerea has a large host range of many different plants, the fungus will grow and sporulate on most of them. Dis- eased tissues of host plants may also contain sclerotia and produce spores. PHOMOPSIS ROT Phomopsis rot of grapes is caused by the fungus Phomopsis viticola Sacc. (fig. 17). The same fungus also causes dead-arm disease of grapevines (Pine, 1948, 1959). In California, Phomopsis rot of grapes generally develops from in- fected cluster stems or pedicels, or both. The fungus grows from the stems into the grape and there causes [46 Fig. 17. Tokay grapes showing Phomop- sis rot caused by Phomopsis viticola Sacc. rot. Rot may also develop from direct infection of grapes by fungus spores during rainy periods around or during harvest time. The fungus develops from small, clear, boat-shaped spores produced in a Mask-shaped structure (pycnidium). In rotting tissues of the grape, pycni- dia are recognized as black erumpent specks in the skin. The beaks of the pycnidia grow out through the skin of the grape (Pine, 1948, 1959). When mature and are wet by rain or heavy dew pycnidia extrude spores in white masses or strings — these are called "spore horns" because they often curl extensively over the beaks of the pyc- nidia (fig. 9). The horns may form from pycnidia in almost any diseased tissue, shoot stems, canes, spurs, leaves, fruit pedicles, etc. Once the horns dis- solve in rain the spores become dis- persed and are spread in drops of water and thus may spread rapidly. The spores infect directly through the skin of grapes of the epidermis of green tis- tues. Incipient infections followed by rains may also develop Phomopsis rots in vineyard or in storage, and/or in the market. Spores that produce rot in storage may also come from pycnidia on infected leaves, canes, and other vine tissues; they may be splashed or washed onto fruit in the cluster before harvest and storage. In California, where many areas have little or no rain during the sum- mer and where humidities are gen- erally low and temperatures high, infection of grape tissues generally occurs in spring. Infection may also take place in the fall at harvest time during rainy periods. Following in- fection in spring, the fungus produces spots in the leaves and blackish-col- ored cankers in shoot-stem tissue, leaf petioles, and in leaf-cluster stems. The fungus grows best at low to moderate temperatures; as temperatures increase in early summer the fungus in cankers will stop growth and remain dormant. In the fall as temperatures drop and dew forms in the evenings, the fungus in some of the black cankers may re- sume growth. It is this fall regrowth in infected tissues in cluster stems that grow into the grapes and causes Pho- mopsis rot. Original infections in fall are generally rare except during rain. The disease has been observed in Emperor, Kandahar, Malaga, Moli- nera, Olivette blanc, Olivette noir, Thompson Seedless, and Tokay. [47] CLADOSPORIUM ROT f.*-M» Fig. 18. Cladosporium rot of Tokay grapes. Upper row shows spots on grapes at time of removal from storage; grape on far left shows slip-skin caused by rubbing with the finger. Lower row shows heavy dark olive-green sporulation and gray fungus growth on surface 5 days after removal from storage. Cladosporium rot occurs periodically on grapes in storage and on late sea- son grapes in the vineyard (fig. 18). The disease is caused by the fungus Cladosporium herbarum (Pers.) Lk. Early signs are small black circular spots under the skin which enlarge slowly to i/9-inch or more in diameter. As the spots grow in storage, the cen- tral portion develops an olive green velvet-appearing fungus mat of hy- phae and spores. Out of storage the mat will enlarge to cover the entire spot. The fungus can infect grapes through uninjured skin. Infection may occur between 38 and 85° F; the opti- mum, however, is 65 to 70° F. Many varieties of grapes are susceptible, but the disease has been observed most frequently in years of early fall rains and on varieties Tokay and Emperor (Delp, etal, 1951). C. herbarum is also one of the or- ganisms that rots raisins. It will de- velop on raisins drying on trays if dew forms, and also on raisins wet by rain. ROT PREVENTION AND CONTROL Specific measures on control and on secondary sources of grape rot orga- appropriate fungicides should be ob- tained from your local Farm Advisor. Vineyards and nearby crops are the primary sources of organisms that rot grapes. Packing, handling, storage, transit vehicles, and market places are nisms. Control measures are based upon (1) preventing development of the diseases by means of cultural prac- tices that will reduce rot organisms in the vineyard, (2) altering other factors that may contribute to the develop- [48] ment of rot, and (3) application of chemicals for protection from infec- tion and rot. In California, the only fungi that infect unwounded grapes directly and cause rot are A. tenuis, B. cinerea, C. herbarum, D. natalensis and P. viti- cola. Most rot fungi infect grapes only through wounds and rot grape tis- sues (the wounds are thus "infection courts" for many rot fungi). Under optimum conditions, species of Rhi- zopus can also spread from grape to grape in a bunch and, given the right conditions, can rot the inside of the cluster. However, Rhizopus fungi do not infect through the unbroken skin of grapes. Fruit flies and dried fruit beetles also transport fungus spores, yeast, and bacteria. When flies and beetles walk into an injury in the grape skin, spores dislodged from them are often left in the injury, where they germi- nate, grow, and cause rot. Sources of inoculum Rot fungi other than Diplodia and Phomopsis usually grow and sporulate on weak or weakened plant tissues or on dying or dead plant parts. Fungus colonies growing on these different plant tissues produce spores, and in some seasons high numbers of spores. A. tenuis and C. herbarum also grow in the bark of canes and spurs, on mummified fruit of old clusters, and in wet springs they sporulate on these surfaces; the spore load can be very high. In cool, wet weather the disease known as "scurf" of shoots develops most abundantly— this disease is caused by infection of tender young shoot- tissues by A. tenuis. B. cinerea overwinters mostly in the form of sclerotia which serve to carry the fungus through unfavorable weather conditions. In high-moisture periods of moderate temperatures, fungus cells in the sclerotia grow and produce conidiospores (special branches of the fungus) on which are borne large numbers of spores. Sclero- tia can be found on old cluster stems and mummified grapes on vines or on the ground under the vines. In vine- yards where Botrytis rot is a problem there are many old cluster stems re- maining, and these have large popula- tions of sclerotia which can produce great numbers of conidia, thus form- ing a high "inoculum potential" (the number of spores present in a vine- yard). B. cinerea also cause blight of many different plants. Long wet periods with temperatures ranging from 45° to 65° F favor development of Botrytis not only on grapes but on all sorts of plant tissues. In some seasons B. cine- ria, A. tenuis, C. herbarum, Stemphyl- lium botryosum and a few other fungi infect and decay grape flower parts, especially old anthers and pollen. Dur- ing bloom in times of cool weather and high relative humidity many of the infected anthers remain in the cluster, and they often remain at- tached to the flower receptacle below the ovary rather than shelling or drop- ping as they normally would. Fungi (often predominantly Botrytis) grow- ing on these flower parts sporulate heavily and add greatly to the inocu- lum. Spores of many of these fungi may also be blown into the vineyard from infected plant parts and debris or from decaying fruit in nearby fields. The number and kinds of spores will vary with the kind of crops, and with conditions in the fields (such as maturity of crop, amount of decay, weather, and general cleanliness). [49] Infection courts Wounds on grapes are common infec- tion courts for most of the grape-rot- ting organisms found in California's vineyards. Fungi having ability to in- fect directly through unwounded skin can also infect through wounds, but they can infect grapes through unin- jured skin only under favorable weather conditions, generally in cool periods of high relative humidity or in free moisture. B. cinerea and D. natalensis infect grapes at bloom time, entering the grape through the style and causing rot in mid-season or when grapes are approaching maturity. In high relative-humidity periods the nec- tar on stigmas of blossom ovaries is generally dilute and thus favorable for fungus-spore germination and growth. Control by prevention Reducing fungus inoculum in the vineyard is a good way of preventing rots of grapes; high inoculum poten- tial can sometimes even override cer- tain chemical control measures. It seems reasonable to believe that al- most any measure that will reduce fungus inoculum, fruit flies, and dried fruit beetles would result in less rot, but experiments to prove this have not been done. Such experiments would require large plots of vineyards (be- cause fungus spores are wind-blown) and such plots are difficult to manage. However, it has been demonstrated that when paper bags were placed over clusters early in the season rot did not develop as much as in the bagged clusters as in unbagged— even in Ribier grapes that had cracked be- cause of faulty water management. Preventing injury to grapes will also result in reduction of rot. Some of the most common injuries to grapes in vineyards are bird pecks, worm dam- age, punctures from bugs (stink bugs), growth cracks about russeted areas, and growth cracks caused by faulty irrigation practices. Diseases such as black measles and powdery mildew also result in cracking of grapes. Spores of rot-causing fungi may be considered to be inoculum; the higher the number of spores, the greater the inoculum potential and the greater the chance for the spores to contact an infection court. Rots often follow rots A little rot in a vineyard will increase the inoculum potential and, if condi- tions are favorable, can cause more rot which in turn adds to inoculum buildup. Furthermore, rot of grapes may tend to increase from one season to subsequent seasons unless some- thing is done to stop inoculum in- crease. Cleaning up all vineyard debris in the spring should help to reduce not only rots but also some other pests. A clean vineyard prior to bloom, and without dust from cultivation during bloom time, will reduce blossom infec- tion by Diplodia and result in much less summer bunch rot. Trellises, which spread clusters, pre- vent clumping, and position fruit for minimum exposure, should result in less fruit damage and rot. Cultural practices Altering cultural practices may favor the development of a disease. There- fore, it is advisable to determine the probable effects of a change: for ex- ample, when growers stopped burning vineyard prunings and began shred- ding or disking prunings into the soil, D. natalensis increased in vineyards and vineyard soils, thus causing ex- cessive amounts of Diplodia cane blight and summer bunch rot. An- other slight change in cultural prac- tices, that of cutting off cane tips just [50 above ground level in summer to keep them out of the soil or from being covered with soil in disking, was found to reduce cane blight to almost nil and to reduce summer bunch rot to a small percentage. In this last example, the inoculum for summer bunch rot came from diseased grape canes disked into the soil; disking in of healthy canes had no apparent effect on build- up of disease. Also, when a long-established cul- tural practice is altered, it may set up a chain of events. For example, chemi- cal weed control in the rows of grape- vines has done away with plowing of the vineyard row, and this permits vineyard trash to accumulate in the rows under the vines. The trash con- sists mostly of cane pieces, leaves, old cluster stems, mummified grapes, and other debris. The old cluster stems and mummified grapes are often in- fected by fungi and usually contain sclerotia of B. cinerea and tissues in- fected with C. herbarum and A. tenuis. Spores of fungi from this debris are wind-blown and add greatly to the inoculum of the vineyard. Obviously, the trash (which also harbor insects) should be destroyed or removed. Chemical control: prevention Fungicides designed to prevent infec- tion by fungi have been used for many years. To be wholly effective, chemi- cals should cover each grape in a cluster— any lesser coverage gives less protection. Most chemicals tested have not given control of individual grape rot nor of bunch rot. Of the chemicals tested, only benomyl for control of early Botrytis rot, and captan when used for control of Botrytis rot asso- ciated with fall rains, have more than paid for the cost of application. Rots resulting from infection through in- juries have not been controlled by chemicals used as dust or sprays. An- alyses of the problem indicates that the only practical control of this form of rot is prevention of the injury. Chemicals for preventing infection at or during bloom time must either be applied several times to obtain cov- erage of blossoms or must be systemic. Chemicals having this property, and also having toxicity for a specific fun- gus (such as B. cinerea), have been experimentally proved to give good control of early Botrytis rot when properly applied at the right time. The chemical control of summer bunch rot initiated by D. natalensis has not been possible, because chemi- cals tested were either not sufficiently toxic to the fungus or were not sys- temic and thus not able to move into the flower and stop infection. Chemicals applied late in the season to control rot must have very low or no mammalian toxicity, and grapes at harvest time must not contain amounts of chemicals even remotely toxic to man. Furthermore, grapes used in wine-making must not carry any chemical control residues into the wine they make. The reader should consult his local Farm Advisor for information on the effective and proper use of chemicals available for the control of rot dis- eases of grapes. REFERENCES Barb, G. D., and W. B. Hewitt 1965. The principal fungus in the summer bunch rot of grapes. Phytopathology 55:815-16. Ciccarone, Antonio 1970. Attuali cognizioni intorno a Botrytis cinerea Pers. sulla Vite. Atti, 22:3-33. 51 Delp, C. J., W. B. Hewitt, and K. E. 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The mold complex of Napa Valley grapes. Amer. J. of Enol. and Vit. 17(2):87-94. McClellan, W. D. 1972. Early Botrytis rot of grapes caused by Botrytis cinerea Pers.: Time of infection and la- tency, and some aspects of control. Ph.D. Thesis, Univ. of Calif., Davis. McClellan, W. D., and W. B. Hewitt 1973. Early Botrytis rot of grapes: Time of infection and latency of Botrytis cinerea Pers. in Vitis vinifera L. Phytopathology 63:1151-57. McClellan, W. D., W. B. Hewitt, P. La Vine, and J. Kissler 1973. Early Botrytis rot of grapes and its control. Amer. J. Enol. and Vit. 24:27-30. Nelson, K. E. 1951 (a). Factors influencing the infection of table grapes by Botrytis cinerea (Pers.) Phyto- pathology 41:319-26. 1951 (b). Effects of humidity on infection of table grapes by Botrytis cinerea. Phytopathology 41:859-64. 1956. The effects of Botrytis infection on the tissue of Tokay grapes. Phytopathology 46:223-29. Nelson, K. E., and M. A. Amerine 1956. 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(Abst.) Phyto- pathology 47:30. Strobel, G. A., and W. B. Hewitt 1964. Time of infection and latency of Diplodia viticola in Vitis vinifera var Thompson Seedless. Phytopathology 54:637-39. Winkler, A. J. 1962. General Viticulture. Berkeley, Calif.: University of California Press. 633 pp. 10m-ll,'74(R9732)VL