UNIVERSITY OF CALIFORNIA PUBLICATION S COLLEGE OF AGRICULTURE AGRICULTURAL EXPERIMENT STATION BERKELEY, CALIFORNIA COTTONY ROT OF LEMONS IN CALIFORNIA BY CLAYTON O. SMITH BULLETIN No. 265 Berkeley, Cal., January, 1916 UNIVERSITY OF CALIFORNIA PRESS BERKELEY 1916 Benjamin Ide Wheeler, President of the University. EXPERIMENT STATION STAFF HEADS OF DIVISIONS Thomas Forsyth Hunt, Director. Edward J. Wickson, Horticulture (Emeritus). Herbert J. Webber, Director Citrus Experiment Station; Plant Breeding. Hubert E. Van Norman, Vice-Director; Dairy Management. William A. Setchell, Botany. Myer E. Jaffa, Nutrition. Eobert H. Loughridge, Soil Chemistry and Physics (Emeritus). Charles W. Woodworth, Entomology. Ralph E. Smith, Plant Pathology. J. Eliot Coit, Citriculture. John W. Gilmore, Agronomy. Charles F. Shaw, Soil Technology. John W. Gregg, Landscape Gardening and Floriculture. Frederic T. Bioletti, Viticulture and Enology. Warren T. Clarke, Agricultural Extension. John S. Burd, Agricultural Chemistry. Charles B. Lipman, Soil Chemistry and Bacteriology. Clarence M. Haring, Veterinary Science and Bacteriology. Ernest B. Babcock, Genetics. Gordon H. True, Animal Husbandry. James T. Barrett, Plant Pathology. Fritz W. Woll, Animal Nutrition. A. V. Stubenrauch, Pomology. Walter Mulford, Forestry. W. P. Kelley, Agricultural Chemistry. H. J. Quayle, Entomology. Elwood Mead, Rural Institutions. J. B. Davidson, Agricultural Engineering. II. S. Reed, Plant Physiology. D. T. Mason, Forestry. William G. Hummel, Agricultural Education. Leon M. Davis, Dairy Industry. John E. Dougherty, Poultry Husbandry. S. S. Rogers, Olericulture. *Frank Adams, Experimental Irrigation. David N. Morgan, Assistant to the Director. Mrs. D. L. Bunnell, Librarian. CITRUS EXPERIMENT STATION DIVISION OF PLANT PATHOLOGY J. T. Barrett H. S. Fawcett C. O. Smith *In co-operation Office of Public Roads and Rural Engineering, U. S. D. A. COTTONY ROT OF LEMONS IN CALIFORNIA* By CLAYTON 0. SMITH The decay commonly known as "cottony rot" or "white mold," has been associated with the lemon industry probably from its infancy, but during the past few years the trouble has so increased in import- ance as to demand special study. The disease is widely distributed in all the lemon-growing sections of California, but is not known to occur on Citrus in Florida. The fungus, Sclerotinia libertiana, causing this decay, is widely distributed on other hosts and probably is present in other lemon-growing countries. The chief loss occurs during the winter and spring months, when the fruit is being stored and cured in tents before being packed for shipment. No estimate of the loss in money value is obtainable, but where it becomes established in a viru- lent form, the loss must be considerable. From the records of one of the lemon houses in California the following data of the amount of cottony rot from January to July, 1911, were obtained. During this time 325 cars were in storage. Of these, 97 cars showed amounts varying from 1 to 23 boxes each affected with cottony rot. A box having this trouble means a large percentage of decaying fruit. The decay is more abundant some years than others and especially so when large quantities of fruit are being stored. This is due largely to the greater difficulty in properly inspecting the tents and in detecting the presence of the fungus before it has caused the decay of a large num- ber of fruits by contact with the first infected ones. The decay on the fruit is chiefly characterized by the mass of white cotton-like mycelial growth that rapidly spreads over the infected lemons. In this growth are to be found black seed-like bodies known as sclerotia, Figs. 1 and 5. By this last characteristic the cottony rot decay can with certainty be distinguished from other lemon rots. In the beginning stages, this decay closely resembles that of the grey mold or Botrytis rot, and at this time the two are often confused in the packing house. In this early stage both of the fungi cause a softening and discoloration of the tissue. The affected tissue, while somewhat softer than normal, is not a typical soft rot, and for some time con- siderable firmness of the tissue may remain. In the advanced stages, * Paper No. 22, Citrus Experiment Station, College of Agriculture, Uni- versity of California, Riverside, California. [237] 238 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION however, the tissue becomes more completely broken down and watery. In the cottony rot as a usual thing the infected tissue is pale lemon in color, while in that of the grey mold it is darker, being almost chocolate. When the aerial growth of the fungi appears, all difficulty in distinguishing between the two decays disappears. In the cottony rot the mycelial growth is white and non-spore producing, but contains hard black sclerotia. In the grey mold (Botrytis) there are numerous spores that can readily be seen in mass. The whole growth and spore masses have a characteristic greyish color. The cottony rot has no dis- tinguishing odor as has the brown rot (Pythiacystis citrophthora) , so Fig. 1. — Sclerotia of cottony rot, as produced under cover crops. From these the peziza or spore stage grows. that no difficulty should be had in deciding between the decays of brown and cottony rot. The twig infection of citrus by the cottony rot is often accompanied with gumming. The infected bark is of a cinerous or ash color and fibrous in its advanced stages. The characteristic sclerotia, Fig. lb, are often, but not always, present. When the twig is at length sur- rounded by the fungus, death takes place suddenly, the leaves become rolled-up and dead, but may remain attached for some months. This last characteristic is the most noticeable and is what usually attracts the grower's attention to this disease in the orchard. COTTONY ROT OF LEMONS IN CALIFORNIA 239 NATURAL INFECTION Picking-Box Infection. — It is only occasionally that the conditions are favorable for cottony rot to develop in the picking boxes, as the fruit remains in these only a short time before being washed. Several instances, however, have come to our notice in which, under favorable conditions, a large amount of this decay (possibly as much as 75 per cent) did develop. The fruit was picked and left out in a rain of several days' duration without protection. Under these conditions not only a great amount of decay was to be detected when the fruit was Fig. 2. — Peziza or spore stage showing varying lengths of stem from sclerotia to the funnel-shaped disk, apothecia, of cottony rot fungus. The length of stem varies according to the depth below the surface of the sclerotium. washed, but this continued to appear during the process of curing. In a few localities the fruit is stored in the picking boxes and no wash- ing of the fruit is considered necessary. In these cases the picking box becomes also the storage box. Packing -House Infection. — The infection of fruit in the packing house is the chief injury done by the cottony rot fungus. The fruit is left in the curing tents or tight rooms from one to two months after being washed, when the final sorting, grading and packing of the fruit takes plaee. During this time, especially on fruit unusually weak, the cottony rot has a favorable chance to develop. The decay is found almost entirely during the spring and winter months, January to 240 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION May, rarely to June. After this time, the decay almost entirely dis- appears. The lemons during curing are kept under rather moist con- ditions to prevent wilting, and in the stacked boxes the conditions are ideal for Sclerotinia to develop. The rot when once started, becomes virulent and a large mass of fruit, even green fruit, may become in- volved (Figs. 4 and 5). The fungus secretes a substance, perhaps an enzyme, that has the power of softening the tissue of a healthy lemon and so prepares the way for the mycelium to enter. When the disease once becomes started in a half box of lemons, almost every lemon soon becomes infected and the decay often spreads upward and downward, extending through the entire stack of fruit. Workers in the packing house have the idea that the drip from the rotting of the fruit into the box containing sound fruit beneath, has much to do with the spread of the fungus through the stack of fruit. This has never been defi- nitely proved, but without question the mycelium does grow through the open spaces in the bottom of the boxes to sound fruit below. Care should be exercised in the handling of contact fruit ; that is, fruit that has been touching cot- tony rot decay. This will be con- sidered in detail later under "con- trol methods." In most respects the treatment is similar to that recommended for brown rot, and with this most packing house work- ers are familiar. Fig. 3. — Section showing how the spores are produced in long sacks or asci that form the spore-bearing sur- face of the f angus. Seed-Bed Infection. — The cottony rot is not regarded as a serious disease of citrus seed-bed stock. It has, however, been found occasion- ally attacking sour and sweet orange seedlings. The shaded moist condition of the seed bed covered with lath or burlap, gives ideal con- ditions for the fungus to thrive. The disease usually shows in small patches of six to a dozen trees. It is thought to gain entrance into sound tissue by starting as a saprophyte on weakened or fallen leaves that are in close contact with healthy leaves or stems. Trees in the seed bed often grow very close together. Seedlings iy 2 to 3 feet high have been found to be attacked, although there is no reason why those smaller or larger might not be infected as easily. COTTONY ROT OF LEMONS IN CALIFORNIA 241 Nursery Infection. — The disease only rarely does any marked in- jury on citrus nursery trees. It usually attacks trees scattered here and there throughout the nursery and shows no evidence of spreading to adjacent healthy ones (Fig. 6). The diseased trees show the same characteristics as do the infected twigs of orchard trees and should be given the same treatment. Orchard Injection. — Among citrus trees the lemon is more often attacked than the orange. The disease appears to be localized on a branch, the infection often occurring some distance from the end of the twigs. Smaller twigs are more often infected, although vigorous growing sprouts are also attacked. The twigs may become infected Fig. 4. — Cottony rot affecting lemons by contact in a packinghouse box. through the blossoms or through some injury. The disease is often worse following a cool winter when the tissue is weakened or injured by frost. Definite infected areas developed on the twig. These increase in size, soon entirely girdling the twig and causing the sudden death of the part beyond the place of infection. The disease does not extend either up or down the infected branch to any great distance. The infected area at first shows a softening of the bark tissue with a con- siderable amount of gumming (Fig. 7a). The bark of the diseased area is often of a lighter color and at length shows a fibrous condition (Fig. 7c). With the appearance of the gumming of the twig the spread of the infection appears to be checked. On an infected twig the leaves remain for a long time in a rolled-up dead condition (Fig. 242 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 8) . Small twigs are more frequently attacked, although vigorous grow- ing sprouts may also be infected. The infection may sometimes start at the end of the twig and spread for a short distance back into the more vigorous wood. In these cases the spread of the disease can often be shown to follow injury from frost of the preceding winter, although other conditions, as unusually long periods of warm, rainy weather in winter or spring, also favor twig infection. The cottony rot fungus has been found occasionally attacking the twigs and trunk of small lemon trees that have been wrapped with corn stalks for winter protection. This fungus has killed young trees that have been planted in the orchard one year from the nursery. A case was observed by Professor H. S. Fawcett in which the fungus had secured a good foot-hold in the trunk and roots of a large lemon tree, which had been weakened by attacks of gummosis. This tree had been wrapped with manure and burlap to keep the tissue from drying out so as better to favor healing, thus affording ideal conditions for fungous growth, and the formation of characteristic sclerotia. Under favorable conditions the blossoms may become diseased, the infection from here spreading into the fruiting twigs. It is believed that blossom infection most often starts on the older petals which, instead of falling, may remain attached in a more or less decadent condition susceptible to the attack of this fungus ( Fig. 9 ) . The blos- soms of lemons often grow in large clusters and a considerable mass of collapsed petals are present where spores can germinate and begin growing as a saprophyte, at length acquiring a parasitic nature capable of infecting healthy tissue. This same fungus at times is a parasite on the blossoms of apricots, 1 attacking them while still enclosed by the calyx or outer cup of the blossom. The leaves of lemons are sometimes attacked while they are at- tached to the tree. There is not, however, any marked defoliation, probably only the older and weaker leaves being susceptible. Cover-Crop Infection. — There is no question but what the cottony rot decay has increased with the increased growing of cover crops. The same fungus not only occurs as a rot of the lemon, but is also capable of becoming a parasite on certain cover crops, vetch being by far the most common host. "While the fungus is common on vetch, the injury it does to the crop as a whole is negligible. The dense growth of vetch affords a suitable place for the fungus to grow as a saprophyte or weak parasite on the weaker shaded portion of the vines. (Fig. 10) Under the vetch cover crop the sclerotia are produced in large numbers i Bulletin No. 218, California Experiment Station, p. 1097. COTTONY ROT OF LEMONS IN CALIFORNIA 243 Fig. 5. — Cottony rot affecting lemons by contact in a packing-house tray. 244 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION and can be found in infested orchards with little searching. The more upright cover crops, like Melilotus indica, would probably be less favor- able for the development of the fungus, although this phase of the subject has not been thoroughly investigated. The spore stage has been found in orchards having a straw mulch, but never in so large numbers as with vetch (Fig. 11). There is no question of a definite relationship existing between this fungus and some of the common cover crops in use, but this apparently should not in any way dis- courage their use. The control of the fungus must be sought for in other ways than through discontinuing the use of green-manure crops. CAUSE OF DISEASE The decay, as already suggested, is due to a fungus belonging to the genus Sclerotinia. From the preliminary study thus far made, it is believed that the fungus is Sclerotinia libertiana, although another species of this genus, probably Sclerotinia trifoliorum, is able to de- velop on lemons from artificial inoculations and possibly may cause some of the infection. Relation to other Hosts. — This fungus is not restricted to citrus for a host, but is an important trouble, attacking various other plants, especially seedlings, certain of the vegetables, green-manure plants, and weeds. The following are some of the hosts known to be affected by the lemon Sclerotinia, or one that closely resembles it pathogenic- ally and morphologically : Lemon fruit, twig and blossoms of lemon, orange twigs, apricot blossoms, rarely avocado twigs and blossoms, lettuce, tomato, alfalfa, ginseng, celery, bean, cucumber, vetch, nettle (Urtica urens) and prickly lettuce (Lactuca scariola), two common weeds in citrus orchards, and occasionally the leaves and weaker stem; of barley and oats. As a saprophyte, the fungus could attack, under favorable conditions, many sorts of dead organic matter and decaying plants. From spore measurements and other microscopic characteristics of the cottony rot fungus, it has always been regarded as the species, libertiana. To determine more fully this point, cultures of Sclerotinia libertiana, were requested from several of the Experiment Stations. Puncture inoculations from these were made in the twigs of Eureka lemon, as well as on the fruit. The results of these are summarized under artificial inoculations, Table I, p. 252, and in almost every case gave definite, positive results. There remains little doubt of the cot- tony rot fungus being identical with Sclerotinia libertiana. The following are the different hosts and localities from which these cultures were secured: (1) Several different cultures from COTTONY ROT OF LEMONS IN CALIFORNIA 245 lemon fruits, California; (2) six or more different strains from lemon twigs, California; (3) alfalfa, California and Oregon Experiment Stations; (4) cucumber, California and Oregon Experiment Stations; (5) lettuce, California and Ex- periment Stations of North Caro- lina, Maine and Wisconsin; (6) eggplant, California; (7) tomato in greenhouse, California; (8) celery, Calif ornia ; (9) bean stem, Maine Experiment Station ; (10) snapdragon, Maine Experiment Station; (11) ginseng, from Cor- nell University; (12) avocado, California. This last fungus was isolated by Professor H. S. Fawcett from twigs of avocado which had been killed from infec- tion that appeared to have start- ed in the blossoms and spread into the more woody twigs. Life History of Fungus. — The life history of the fungus consists of three stages, differing in appearance and functions : (1) The vegetative stage is the white cotton-like growth so com- mon in the packing houses. This growth has no spores, but can spread rapidly and has the power to infect sound fruit simply by coming in contact with and spreading over it. (2) In this white growth, numerous black bodies known as sclerotia are de- veloped (Fig. 1). These are nothing more than a very solid dense mass of mycelium, so form- ed that they will resist long periods of hot dry weather or the cold freezing conditions of the eastern states. After these bodies have gone through a period of rest and the favorable rainy, cool weather of the autumn and winter Fij . — Eureka lemon tree in nursery affected with cottony rot. 246 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION has come, they revive and become active. The sclerotia may then send out a fresh growth of mycelium, the vegetative stage, which could easily infect dead organic matter or the weaker plant growth of weeds Pig. 7. — Cottony rot affecting the twigs of a Eureka lemon tree, (a) Masses of gum produced by the fungus, (b) Sclerotia of the fungus distinguished from the gum globules by more spherical shape, (c) Fibrous condition of infected bark. or cover crops. Here after a time new sclerotia are again formed. (3) From the sclerotia in the soil the spore stage is formed under the cover crops in the lemon orchards. This last is accomplished by sending out a growth which eventually develops into a small funnel- COTTONY ROT OF LEMONS IN CALIFORNIA 247 shaped, cup-like disk, technically called an apothecium (Fig. 2). In the flesh of the apothecia are produced the spores. These are formed in rather long cylindrical sacks or asci (Fig. 3). Hence the spores are called ascospores. Both the spores and asci are microscopic. The spores are easily scattered by the wind. The fungus also has the power to force out or discharge these spores, and they can often be observed leaving the apothecium as small clouds of dust. Some of Fig. 8. — Artificial inoculations of twigs of Navel orange. Note the rolling of leaves which have become dead and bleached to almost a white color and yet still remain attached to branch. these spores are carried by the wind to the fruit and twigs, of the lemon and cover crops, where under favorable conditions they ger- minate and produce the vegetative stage. The apothecial stage in California begins to appear about the first of October and continues to be formed during the winter and spring. After the cover crops of the orchards are plowed under and cultiva- tion begins, few apothecia (spore stage) are thought to be produced, for this stage of the fungus requires moist, shaded conditions, such as are best found under cover crops during the rainy season. 248 UNIVERSITY OP CALIFORNIA — EXPERIMENT STATION ARTIFICIAL INOCULATION EXPERIMENTS On Fruit. — The disease can be readily produced on the ripe or green lemons when left in a moist chamber: (1) By placing the mycelium or sclerotia in the tissue through a puncture or injury; (2) by plac- ing mycelium on the uninjured surface of the fruit — this is especially virulent if some other dead organic matter is also present; (3) by placing a piece of an apothecium in the tissue through a puncture or injury ; (4) by atomizing spores on the surface, in which case infection occurs only at the button and blossom ends and at points in contact with each other or with the moist chamber; (5) by inoculating the stem and blossom ends with mycelium of the fungus, infection devel- oped in two weeks' time. Apothecia placed in drops of sterilized distilled water on the surface of lemons gave negative results. Many spores must have been lib- erated in the water, but the germ tubes were not able to infect the un- injured rind. This observation agrees with those of former investi- gations that the mycelium of this fungus needs to grow for a time as a saprophyte before it becomes actively parasitic. The reason for this requirement is perhaps explained by the fact that the mycelium secretes an enzyme or substance that apparently plays a very import- ant part in the softening and breaking-down of the tissue, thus pre- paring the way for its invasion with the fungous hyphae. Spores placed in drops of water or in drops of prune juice give negative results on uninjured tissue. Typical experiments in inoculating lemons with cottony rot. From these and numerous other experiments conclusions as given under artificial inoculations were largely derived : 1. Experiments in atomizing lemons in a moist chamber with spores of the fungus. This is only one of many made during the investigation. The others, however, show practically the same results. (a) In this experiment an attempt was made to imitate as nearly as pos- sible natural conditions. On February 27th, 1911, five nearly ripe lemons were secured from an orchard that had always been free from this decay. The fruit was picked during a rainy period beginning on February 25th and con- tinuing more or less until March 2d. The fruit w T as first sterilized for five minutes in 4 per cent formalin* and then rinsed in tap water and left out in the rain for a short time before being atomized with spores. The fruit was then placed in a quart fruit jar for a moist chamber, and left out in the rain where the temperature was somewhat cooler than that in the laboratory. When the rain ceased the jar was covered with a large plant pot to exclude the light. In five days the fruit was examined and found to be infected at * An aqueous solution of 40 per cent formaldehyde is a commercial product, and is called ''Formalin". COTTONY ROT OP LEMONS IN CALIFORNIA 249 all points where it was in contact. Four of the five showed infection at the stem end. At this time there was no evidence of aerial mycelial growth, but simply a small amount of tissue infected and softened at all points where the fruits were touching each other and at the stem end. Characteristic mycelium and sclerotia developed later. (b) Secured from lemon orchard fruit showing the small beginning spot of brown rot (Pythiaeystis citrophthora) . The fruit at this time is only slightly pitted or depressed at point of infection. Sterilized the brown rot fruit in 4 per cent formalin for five minutes and rinsed same in tap water. No injury by germicide was observed on these lemons. On February 25, 1911, this fruit was atomized with spores of cottony rot. On March 8th, the brown rot had spread over about one-third of the surface of the lemons. The cottony rot was also developing over the brown rot areas and had invaded some of the tissue not yet infected by the brown rot fungus. On March 14th, the cottony rot had completely overgrown and covered the entire fruit. The cottony rot seems to attack readily tissue in which the brown rot has become established, in brown rot fruit kept as a check without being inoculated with cottony rot spores, no cottony rot appeared. 2. Experiments in soaking lemons in water inoculated with spores of cottony rot. Experiments were made at several different times following in a somewhat limited way methods similar to those used in inoculating lemons with the brown rot. A receptacle of sufficient size to contain twelve lemons was filled with sterile water inoculated with macerated apothecia of cottony rot. On Decem- ber 28, 1910, soaked lemons in this solution for 36 hours, and at the end of two weeks' time there was no evidence of decay. February 3, 1911, the experi- ment was repeated with thirteen green lemons soaked in tap water mixed with six large apothecia secured from under a vetch cover crop. The apothecia were first macerated in small pieces, and the fruit was left for 24 hours. On Marcli 29, 1911, the fruit show r ed no evidence of decay. 3. Experiments in inoculating the fruit in moist chamber through punctures and other mechanical injuries. This method rarely fails of giving positive results. February 15, 1911, inoculated three lemons in moist chamber with pieces of the apothecia, first making a slit in the rind of fruit on side and at stem end. These apothecia came from two different sources: (1) from the orchard under a vetch cover crop; (2) from cultures isolated from a diseased orange branch. February 25, 1911, all the inoculated lemons showed infection at the points of inoculation. 4. Experiment in inoculating fruit by placing mycelium on the uninjured surface. (a) December 28, 1910, inoculated stem or button end of three lemons with mycelium taken from moist chamber where the cottony rot fungus was grow- ing on a lemon. January 15, 1911, slight indication of softening of tissue around stem. February 16, 1911, area about stem had softened in every case and later developed typical cottony rot. (b) December 22, 1910, placed mycelium from a cottony rot lemon on the uninjured surface of five lemons. January 3, 1911, showed diseased, softened areas about an eighth of an inch in diameter in each place where the surface was inoculated. 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