UNIVERSITY OF CALIFORNIA 
 COLLEGE OF AGRICULTURE 
 AGRICULTURAL EXPERIMENT STATION 
 BERKELEY, CALIFORNIA 
 
 ALTERNARIA ROT OF LEMONS 
 
 E. T. BARTHOLOMEW 
 
 BULLETIN 408 
 
 October, 1926 
 
 UNIVERSITY OF CALIFORNIA PRINTING OFFICE 
 
 BERKELEY, CALIFORNIA 
 
 1926 
 
Digitized by the Internet Archive 
 
 in 2012 with funding from 
 
 University of California, Davis Libraries 
 
 http://www.archive.org/details/alternariarotofl408bart 
 
ALTERNARIA ROT OF LEMONS 
 
 E. T. BARTHOLOMEW 2 
 
 INTRODUCTION 
 
 With the possible exception of Penicillium, Alternaria is probably 
 instrumental in causing more California lemons to decay than any 
 other known fungus. That the same may be true for other lemon- 
 producing localities is indicated by the following quotation: "In six 
 cars of lemons from Italy, from 4 to 55% or an average of 18% of 
 Alternaria-inf ected lemons was found. ' ' 3 The same article states that 
 while this survey was being made ninety-one cars of lemons from 
 California showed an average of 10 per cent of Alternaria rot. 
 
 In this connection it is of interest that Fawcett 4 found, as a result 
 of a study of the lemons shipped to the central and eastern markets of 
 the United States during the latter part of July and in August, 1924, 
 that under the conditions prevailing at that time Alternaria was 
 responsible for more decay than all of the other fungi combined. 
 
 Alternaria rot develops in lemon fruits almost exclusively under 
 storage, transit or market conditions. It appears in the unpicked fruit 
 only when it is allowed to become over-ripe or when some abnormal 
 condition, such as a freeze, has materially weakened the fruit. As 
 will be shown later on in this paper, hundreds of tests have proved 
 that practically every fruit is potentially infected by the time it is a 
 few weeks old ; but the development of the fungus and the destruction 
 of the fruit is delayed by the vitality of the fruit. 
 
 During the early part of this study decaying fruits were obtained 
 from a large number of the lemon packing houses in southern Cali- 
 fornia and a few from the central and northern parts of the state. 
 These fruits were supposed to be infected with Alternaria and cultural 
 tests confirmed the supposition. The results of these tests led to the 
 conclusion that the fungus is prevalent in all lemon-growing districts 
 of the state. While subsequent observations have confirmed this, it 
 
 1 Paper No. 149, University of California, Graduate School of Tropical Agri- 
 culture and Citrus Experiment Station, Eiverside, California. 
 
 2 Associate Plant Pathologist in the Experiment Station, Citrus Experiment 
 Station, Riverside, California. 
 
 3 Anderson, H. W. Plant Disease Survey Supplement 14: 111. 1921. 
 (Reported by the Inspector for the U. S. D. A. Bureau of Markets.) 
 
 4 Fawcett, H. S. The decay of citrus fruits on arrival and in storage in 
 eastern markets. Calif. Citrograph 10: 79, 98-99, 103. 1925. 
 
4 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 was observed also that the fruit from certain districts or from indi- 
 vidual packing houses showed more decay than that from other sources. 
 This is probably due to variations in the vitality of the fruit or else 
 to differences in the packing house conditions. It cannot be too 
 strongly emphasized that any storage, transit or market condition that 
 tends to weaken the fruit will also tend to promote the development 
 of Alternaria, since this fungus usually develops very slowly, if at 
 all, in lemons of high vitality. 
 
 A systematic study of this species of Alternaria has not been made. 
 However, its behavior in artificial cultures indicates that at least two 
 forms or strains, if not species, may produce the decay in question. 
 In some respects the resemblance to Altemari citri, which causes black 
 rot of the navel orange, is striking, but whether the same organism is 
 involved in the two cases has not yet been determined. 
 
 In the eastern markets especially, one type of this diseased condi- 
 tion of the lemon is referred to as "center rot," because of its char- 
 acteristic invasion of the internal tissues before it is evident on the 
 surface of the fruit. However, there are other fungi which cause a 
 similar decay of the lemon and for this reason it seems best to use here 
 the term "Alternaria rot" rather than the less specific term, "center 
 rot." 
 
 ALTERNARIA ROT AND ENDOXEROSIS COMPARED 
 
 The terms Alternaria rot and endoxerosis 5 should not be used inter- 
 changeably. Until 1920 the decay in lemon fruits, now known as 
 Alternaria rot, was considered by the growers and packers to be the 
 advanced stages of endoxerosis. At that time an intensive study of 
 the disease was begun. Boxes of yellow, silver, and green lemons, half 
 of each lot being sound and the other half affected with endoxerosis, 
 were carefully selected and placed under the usual storage conditions 
 in each of five packing houses. Once every month for four months 
 a portion of the fruit in each lot was cut to detect any increase in 
 percentage of lemons showing endoxerosis and any evidence of its 
 increase in severity in those lemons affected when placed in storage. 
 Any of these lemons which showed a more marked breaking down of 
 the tissues, either externally or internally, as well as samples of those 
 which had remained the same, were brought to the laboratory where 
 cultures were made from the tissues. 
 
 s Endoxerosis (pronounced "en-do-zer-6-sis," means internal drying) is a 
 technical term used in place of the less definite terms locally applied to this 
 malady, such as "internal decline," "blossom-end decay," "dry tip," and 
 "yellow tip." 
 
BULL. 408] ALTERNARIA ROT OF LEMONS 5 
 
 The observations in the packing* houses and the results of the 
 cultural tests appeared to justify the following conclusions: (a) the 
 healthy fruits did not become affected with endoxerosis, (6) those 
 already affected with endoxerosis did not show any increase of the 
 characteristic symptoms, (c) in the cultures no fungus could be 
 isolated from the healthy fruits or from those having only the 
 symptoms of endoxerosis as it appears in the fruits taken directly 
 from the trees, and (d) the cultures showed that actual decay of the 
 healthy lemons and of those having endoxerosis was caused in every 
 case by a fungus. In almost every case Alternaria was found to be 
 the cause of the decay except, of course, where molds had gained 
 entrance through mechanical injuries due to handling. In only a few 
 isolated cases were such fungi as Colletotrichum and Phomopsis found 
 to be the principal causes of the decay. 
 
 In 1921 a similar experiment was conducted in collaboration with 
 15 lemon association packing houses. The houses were widely distri- 
 buted over the lemon-growing districts of southern California. After 
 the experiment was started the individual tests were put into the care 
 of the manager or foreman, or both, of the respective packing houses 
 with the instruction (a) that they make the observations, (&) that 
 they send fruits to the laboratory for making cultures, and (c) that 
 upon the termination of the test they report their conclusions. 
 
 The results of the cultural tests were identical with those made in 
 1920. The breaking down of the tissues of those fruits affected with 
 endoxerosis did not progress, unless they were infected by Alternaria. 
 Reports from thirteen of the packing house managers or foremen 
 showed that no sound lemons became affected with endoxerosis after 
 they were placed in storage. Of the other two, one reported incon- 
 clusive results and one reported that "The decline (endoxerosis) con- 
 tinued to advance in the affected fruit resulting in a final complete 
 drying-up or slushy rot." This last statement does not in any way 
 contradict the conclusion that endoxerosis does not progress in the 
 fruit after it is taken from the tree because the symptoms mentioned 
 are those of Alternaria and not of endoxerosis. The statement serves 
 to indicate the confusion previously existing as to the identity of the 
 two diseases. To prevent future confusion it may be well at this time 
 to state the characteristic symptoms of these two maladies. Plate 1 
 illustrates many of the characteristic differences between these two 
 diseases. 6 
 
 6 See also Fawcett, H. S., and H. A., Lee. Citrus diseases. McGraw-Hill 
 Book Co., N. Y. (For Alternaria rot see fig. 130, A to D, for endoxerosis see 
 fig. 149.) 
 
UNIVERSITY OF CALIFORNIA— EXPERIMENT STATION 
 
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Bull. 408] 
 
 ALTERNARIA ROT OF LEMONS 
 
 The results of these two sets of experiments, performed in 1920 and 
 in 1921, together with widespread observations since that time, have 
 shown conclusively that the breakdown and decay of the fruits in 
 question is due to the action of the fungus Alternaria and not to the 
 so-called " later stages" of endoxerosis. 
 
 INOCULATION EXPERIMENTS 
 
 During the course of the preceding observations and experiments 
 the question arose as to whether the lemon fruits became infected with 
 Alternaria before or after being picked. An effort was made to 
 answer this question by making inoculation experiments first in two 
 packing houses and later under controlled conditions in the laboratory. 
 
 TABLE 1 
 
 Inoculation Experiments in the Packing House. Comparative Percentages 
 of Alternaria Rot Appearing in Inoculated and Uninoculated Sound and 
 Endoxerotic Lemons after 90 Days in Storage. 
 
 
 Proportion 
 
 of lemons 
 
 affected with 
 
 endoxerosis 
 
 Lemons infected by Alternaria 
 
 Average 
 
 Condition of lemons 
 
 Inoculated 
 
 Checks 
 
 percentage of 
 Alternaria 
 
 Yellow, sound 
 
 Per cent 
 
 20 
 75 
 15 
 50 
 10 
 15 
 
 Per cent 
 
 17 
 35 
 
 9 
 20 
 
 5 
 10 
 
 Per cent 
 
 6 
 25 
 
 9 
 30 
 
 3 
 
 6 
 
 Per cent 
 12 
 
 Yellow, endoxerotic... 
 
 30 • 
 
 Silver, sound 
 
 9 
 
 Silver, endoxerotic 
 
 Green, sound 
 
 Green, endoxerotic 
 
 25 
 
 4 
 
 8 
 
 In the packing houses. — Two hundred lemons of each of the kinds 
 indicated in table 1 were taken from the washer and dried. Before 
 these fruits were placed under the usual storage conditions, those 
 designated as "inoculated" were "painted" with a suspension of 
 Alternaria spores, usually on the stem end around the button but in 
 some cases on both stem and stylar ends. At the end of each of three 
 30-day intervals the percentages of Alternaria appearing in each lot 
 of fruit were determined (table 1). The fruit in one packing house 
 showed a slightly higher percentage of Alternaria in each lot than that 
 in the other house, but the ratios between the different kinds of fruit 
 were similar. 
 
 The results are not conclusive. They show a higher percentage of 
 Alternaria rot in the inoculated yellows and greens but not in the 
 silvers. While the results show that in four of the six lots the amount 
 
8 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 of Alternaria rot appearing within this three-month period was 
 apparently increased through inoculation, yet they clearly do not 
 prove that none of the lemons in any of the lots would have decayed 
 without inoculation. The results of some of the experiments to be 
 described a little farther on in this paper will give additional evi- 
 dence on this question. No infections resulted from the stylar-end 
 inoculations. 
 
 A point of particular interest in connection with this experiment is 
 the fact that a much larger percentage of Alternaria rot appeared in 
 the endoxerotic than in the sound lemons, as indicated in the last 
 column of table 1. These and other experiments and observations 
 indicate that this was due to the lower vitality of endoxerotic lemons, 
 which makes them less able to resist invasion by the fungus. 
 
 In the laboratory. — The inoculation experiments in the laboratory 
 were confined to silver and light-green lemons. They were carefully 
 sorted and only those of the best quality were used. Each group of 
 silvers and light greens was divided into twenty-one lots of twenty 
 lemons each. Of the twenty-one lots of silvers seven were used as 
 controls, seven were soaked five minutes in copper sulphate solution 
 (%o of 1 per cent, as commonly used in the lemon washer in the 
 packing houses) and the other seven, after soaking as in the preceding 
 case, were dried and then inoculated with a suspension of a pure 
 culture of Alternaria spores in water. The suspension was applied by 
 means of a camel's-hair brush and, in so far as possible, was applied 
 under as well as around and on top of the buttons. The twenty-one 
 lots of light greens were divided and treated in the same manner. All 
 lemons were wrapped in standard wrappers. The lots of silvers and 
 light greens were then divided into seven groups in such a manner 
 that each group contained one lot of silvers for controls, one that had 
 been soaked, one that had been soaked and then inoculated and three 
 similar lots of the light greens. Each group therefore included sixty 
 silvers and sixty light greens. One of these seven groups was then 
 placed in each of the seven compartments of a constant temperature 
 apparatus. The respective temperatures of these compartments were 
 approximately 48°, 59°, 68°, 75°, 82°, 90° and 97° F. The tempera- 
 ture in any one compartment did not vary more than one or two 
 degrees during the course of the experiment. The relative humidity 
 in each compartment was at or near the saturation point. At intervals 
 of from one to three weeks for a period of three and a half months the 
 fruit was examined and the number in each lot that showed visible 
 signs of decay was recorded and cultures made, when necessary, to 
 identify the fungus causing the decay. A summary of the results of 
 this experiment is shown in table 2. 
 
Bull. 408] 
 
 ALTERNARIA ROT OF LEMONS 
 
 TABLE 2 
 
 Effect of Attempted Sterilization and of Artificial Inoculation on the 
 Amount of Alternaria Rot Appearing in Sound Silver and Light-green 
 Lemons, under Controlled Temperature Conditions. 
 
 Kind of lemons 
 
 Treatment 
 
 Per cent Alternaria rot at the following 
 temperatures, °F 
 
 Average 
 percentage 
 
 
 48 
 
 59 
 
 68 
 
 75 
 
 82 
 
 90 
 
 97 
 
 Alternaria 
 rot 
 
 Silvers 
 
 Checks 
 
 Soaked in CuS0 4 ... 
 | Soaked in C11SO4, 
 inoculated 
 
 
 
 10 
 
 15 
 
 
 
 10 
 
 20 
 
 5 
 25 
 
 15 
 
 10 
 20 
 
 30 
 
 40 
 40 
 
 40 
 
 50 
 35 
 
 50 
 
 20 
 15 
 
 20 
 
 18 
 22 
 
 27 
 
 Greens 
 
 Checks 
 
 J Soaked in CUSO4.... 
 Soaked in CuS0 4 , 
 inoculated 
 
 
 
 
 
 
 5 
 
 
 5 
 
 
 5 
 
 
 
 10 
 15 
 
 5 
 
 45 
 35 
 
 55 
 
 25 
 20 
 
 35 
 
 25 
 25 
 
 35 
 
 16 
 14 
 
 19 
 
 
 
 
 The results of this experiment show that : 
 
 1. The copper sulphate solution had no sterilizing effect on the 
 lemons, in so far as preventing development of Alternaria rot was 
 concerned. 
 
 2. Artificial inoculation of the lemons apparently increased to a 
 limited extent the percentage of Alternaria rot appearing in the 
 lemons, within the time limits of the experiment, as was also shown 
 in table 1. However, the data in table 3 should be taken into con- 
 sideration in this connection. It may be said also that the water 
 added at the time of making the inoculation was probably an important 
 factor in increasing the amount of decay in the inoculated lemons; 
 the added water increased the amount of moisture under the buttons 
 and thus made conditions more favorable for the growth of the 
 Alternaria. 
 
 3. The optimum temperature for the development of Alternaria in 
 these lemons proved to be in the neighborhood of 82° F. Subsequent 
 experiments and observations have shown that this is approximately 
 the optimum temperature for the development of Alternaria rot in 
 lemons in general. 
 
 On the date of the last examination all of the lemons were removed 
 from the constant-temperature apparatus. A large number of them 
 still showed no exterior indications of decay. This was especially 
 true of those that had been kept at the lower temperatures. As the 
 lemons were removed from the compartments each was cut transversely 
 just below the button to detect any case of Alternaria rot in the early 
 
10 
 
 UNIVERSITY OP CALIFORNIA EXPERIMENT STATION 
 
 stages of development. The percentage in each compartment showing 
 the development of Alternaria is given in table 3. The extent of 
 invasion of the Alternaria, from the buttons into the central tissues 
 of the lemons, ranged from about an eighth to three-quarters of 
 an inch. 
 
 TABLE 3 
 
 Percentages of the Externally Sound Lemons of Table 2, which, when 
 Removed from the Compartments of the Constant -Temperature Appa- 
 ratus and Cut Across just Below the Buttons, Showed that Alternaria 
 had Begun to Invade the Tissues. 
 
 
 Per cent silvers having 
 Alternaria rot 
 
 Average 
 
 Per cent greens having 
 Alternaria rot 
 
 
 Temperature 
 °F 
 
 Controls 
 
 Soaked 
 in CuS0 4 
 
 Soaked 
 in CuSO*, 
 inoc- 
 ulated 
 
 Controls 
 
 Soaked 
 in CuS0 4 
 
 Soaked 
 inCuSO*. 
 inoc- 
 ulated 
 
 Average 
 
 97 
 
 100 
 
 100 
 
 100 
 
 100 
 
 100 
 
 100 
 
 100 
 
 100 
 
 90 
 
 100 
 
 100 
 
 100 
 
 100 
 
 98 
 
 96 
 
 99 
 
 98 
 
 82 
 
 100 
 
 98 
 
 99 
 
 99 
 
 91 
 
 89 
 
 88 
 
 89 
 
 75 
 
 100 
 
 91 
 
 92 
 
 94 
 
 78 
 
 75 
 
 78 
 
 77 
 
 68 
 
 79 
 
 69- 
 
 76 
 
 75 
 
 70 
 
 79 
 
 60 
 
 70 
 
 59 
 
 55 
 
 72 
 
 69 
 
 65 
 
 42 
 
 45 
 
 42 
 
 43 
 
 48 
 
 55 
 
 61 
 
 59 
 
 58 
 
 35 
 
 45 
 
 40 
 
 40 
 
 Average.... 
 
 84 
 
 84 
 
 85 
 
 84 
 
 73 
 
 76 
 
 72 
 
 74 
 
 The data in table 3 show : 
 
 1. A large percentage of the apparently sound lemons was found 
 to be already infected with Alternaria in the early stages. This was 
 especially true of the higher temperatures. The fact that at the tem- 
 peratures of 97° and 90° F a larger percentage of the lemons showed 
 infection than at 82° F, which has been given as the approximate 
 optimum for the development of Alternaria. in lemons, might be taken 
 to indicate that 82° F is really not the optimum. However, the slightly 
 greater percentage of infection at the highest temperatures, which 
 are above the optimum for the growth of the fungus, may have been 
 due to a weakened condition of the lemon tissues resulting from 
 exposure to these high temperatures. Examination showed that the 
 fungus had developed much farther in the lemons held at 82° F than 
 in those held at the higher temperatures. 
 
 2. Averaging the data for all temperatures, the uninoculated silvers 
 had only one per cent less infection than the inoculated, while in the 
 light greens the two groups of uninoculated had one and four per cent 
 
Bull. 408] 
 
 ALTERNARIA ROT OF LEMONS 
 
 11 
 
 respectively more infection than the inoculated. This indicates that 
 the Alternaria organism was already present in or beneath each lemon 
 button when the lemons were picked and with sufficient time and 
 favorable conditions practically all of these lemons would have shown 
 decay without artificial inoculation. 
 
 3. The infection was approximately ten per cent greater in the 
 silvers than in the light greens, showing, as was indicated in tables 1 
 and 2, that the longer the lemons are left on the trees after becoming 
 practically mature, the sooner will they show decay by Alternaria 
 after being placed in storage. The data in table 5 emphasize the 
 correctness of this statement even more markedly. 
 
 GROWTH RATE OF ALTERNARIA MYCELIUM 
 
 Having found that 82° F was approximately the optimum tem- 
 perature for the development of Alternaria rot in the lemons, it seemed 
 advisable to determine next the optimum temperature for the rate of 
 growth of Alternaria in artificial cultures. For this purpose the same 
 constant-temperature apparatus was used but, as indicated in table 4, 
 
 TABLE 4 
 
 The Effect of Different Temperatures on the Rate of Growth of Alter- 
 naria Mycelium on an Artificial Culture Medium (Cornmeal Agar). 
 Transfers were Made on January 6, at 9 A.M. 
 
 
 Diameters of mycelial discs growing at the following temperatures, °F 
 
 Times of measurement 
 
 48 
 
 58 
 
 66 
 
 72 
 
 79 
 
 85 
 
 94 
 
 Jan. 8, 9 a.m 
 
 Jan. 9, 9 a.m 
 
 Jan. 10, 9 a.m 
 
 cm 
 
 0.4 
 0.5 
 0.9 
 
 cm 
 
 0.8 
 1.7 
 1.9 
 
 cm 
 
 1.6 
 2.3 
 3.0 
 
 cm 
 
 1.6 
 
 2.8 
 3.6 
 
 cm 
 
 2.1 
 3.3 
 5.2 
 
 cm 
 
 2.0 
 3.0 
 4.2 
 
 cm 
 
 0.7 
 0.9 
 1.2 
 
 
 
 the range of temperatures was a, little lower than in the preceding 
 experiment. Sterilized Petri dishes and glucose-potato and cornmeal 
 agar were used. A very small droplet of water containing Alternaria 
 spores in suspension was placed on the center of the culture medium 
 in each Petri dish by means of a small, sterilized platinum loop. An 
 equal number of dishes of the two kinds of culture media was then 
 placed in each of the different constant-temperature compartments. 
 It is characteristic of Alternaria mycelium, when grown on culture, 
 media of this kind, to grow approximately equally in a plane in all 
 directions from the original point of inoculation, thus making a disc- 
 shaped mat. The rate of growth was determined by measuring at 
 
12 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION 
 
 intervals the diameter of each disc of mycelium. While the character 
 of the mycelium on the glucose-potato agar was a little different from 
 that on cornmeal agar, the rate of growth on the two was so nearly 
 the same that only the averages for the latter are given (table 4). 
 
 The data show (a) that the optimum temperature for the growth of 
 Alternaria on these two artificial culture media is approximately the 
 same as in lemon fruits (82° F), and (&) that Alternaria will grow 
 slowly at least, at temperatures as low as 48° and as high as 94° F. 
 These results agree very well with the data obtained on the develop- 
 ment of Alternaria in lemon tissues (table 3). 
 
 RESISTANCE OF ISOLATED ALTERNARIA SPORES AND MYCELIUM 
 
 Tests on the resistance of Alternaria spores and mycelium to 
 mercuric chloride and to hot water were made, preliminary to test- 
 ing the efficiency of various sterilizing solutions for the prevention 
 of Alternaria rot in lemons. 
 
 Mercuric chloride, — Pure cultures of Alternaria, isolated from 
 lemons, were grown on nutrient agar in Petri dishes. As soon as the 
 surface of the medium became well covered with mycelium and spores, 
 small pieces of the agar (approximately %e X Vie X % inch) were 
 removed and placed in small glass funnels lined with filter paper. 
 The funnels were then immersed in a solution of 1 part mercuric 
 chloride to 1000 parts of water and kept at a temperature of 75° F. 
 After periods of 2, 4, 6, 8, and 10 minutes respectively a given number 
 of the funnels was removed from the sterilizing solution and washed 
 repeatedly with sterile distilled water for half an hour to remove all 
 traces of mercuric chloride. The pieces of agar, bearing the spores 
 and mycelium, were then transferred to fresh culture medium in Petri 
 dishes and incubated at 77° F. Small pieces of the original culture 
 which had not been sterilized were washed with sterile distilled water 
 and similarly incubated to serve as checks. 
 
 All of the unsterilized cultures showed the characteristic develop- 
 ment of Alternaria while in all of the treated cultures growth was 
 entirely lacking. 
 
 The results of this experiment show that Alternaria spores and 
 mycelium, grown on artificial culture media, may be killed by immers- 
 ing them for only two minutes in a solution of 1 to 1000 mercuric 
 chloride and water, kept at 75° F. 
 
 In another experiment mature fruits and twigs were brought to 
 the laboratory and their surfaces washed with a brush dipped in sterile 
 distilled water. Portions of the water were then filtered through 
 
BULL. 408] ALTERNARIA ROT OP LEMONS 13 
 
 filter paper in the small funnels used in the previous experiment. 
 Microscopical examination showed the presence of an abundance of 
 Alternaria spores and some bits of mycelium. The mycelium, where 
 present, was composed of small, irregular pieces such as one would 
 expect to find produced under adverse conditions. The process of 
 sterilization was the same as in the preceding experiment. After 
 washing, small masses of the spores and mycelium were scraped from 
 the filter papers, transferred to culture dishes and incubated at 77° F. 
 
 The results were similar to those of the preceding experiment. The 
 unsterilized checks showed 100 per cent growth but there was no 
 growth in any of the cultures where the spores and mycelium had 
 been exposed to the sterilizing solution for periods of 2, 4, 6, 8, or 10 
 minutes. Evidently the Alternaria organism whether obtained from 
 artificial or natural cultures may be killed by immersion for a period 
 of only two minutes in a solution of 1 part mercuric chloride in 1000 
 parts of water at a temperature of 75° F. 
 
 Hot water. — Since hot water at a temperature of 115 to 125° F 
 is commonly used in the lemon packing houses as a means of controlling 
 brown rot (Pythiacystis citrophthora) , it seemed desirable to deter- 
 mine the effects of hot-water treatment on Alternaria, This test was 
 conducted in the same manner as the two preceding ones, except that 
 hot water (116° F) was used as the sterilizing medium instead of 
 mercuric chloride. 
 
 In this test all cultures showed 100 per cent Alternaria infection. 
 It was interesting to note, however, that the treatment retarded the 
 rate of growth, especially in the cultures that had been exposed for 
 ten minutes. 
 
 RESISTANCE OF ALTERNARIA SPORES AND MYCELIUM WHILE 
 
 ADHERING TO THE SURFACE OF LEMON 
 
 FRUITS AND TWIGS 
 
 The mercuric chloride solution was tested next on spores and 
 mycelium in contact with the surface of the tissues. Thin strips of 
 peel taken from mature lemon fruits were first used. Microscopical 
 study showed that the mycelium had not penetrated the surface layer 
 of cells. The strips, approximately % inch thick, were cut into pieces 
 about % inch square. Forty of these pieces were put into each of 
 five cheese-cloth bags, and vacuumized in a flask of water for one hour 
 to remove all air that might prevent the sterilizing solution from 
 coming into contact with the spores or mycelium. Upon removal from 
 the vacuum flask four of the bags were immersed in the sterilizing 
 
14 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 solution. They were removed successively from this solution at 
 intervals of 2, 4, 6, and 8 minutes. After thorough washing to remove 
 the mercuric chloride, the pieces of peel were plated on a culture 
 medium and incubated at 77° P. The pieces of peel from the fifth 
 bag were not sterilized but were cultured in the same way as the others, 
 to serve as checks. The sterilizing solution was the same as before 
 and was used at 73° P. 
 
 The percentages of Alternaria appearing in these cultures at the 
 end of eight days were as follows: checks, 35 per cent; immersed 
 2 minutes, 18 per cent ; 4 minutes, 13 per cent ; 6 minutes, 8 per cent ; 
 and 8 minutes, none. 
 
 The second part of the test was similar to the first part except that 
 portions of twigs were used instead of pieces of peel,, and they were 
 immersed in the sterilizing solution for only two minutes. The 
 portion of the twig used was about % inch of the pedicel, the 
 portion of the stem just below the button. This part of the twig is 
 free from bracts, which might cover the spores, and to the unaided eye 
 it appears to be entirely smooth. Sixty-four of these pieces were 
 treated and then cultured as in the first part of the test. 
 
 As in the case of the peel it was found that a two-minute exposure 
 to the sterilizing solution was not sufficient to kill all of the Alternaria, 
 After one week in the incubator 19 per cent of the pieces showed a 
 growth of Alternaria. 
 
 The results of both parts of the preceding test indicate rather 
 strongly that even though an exposure of isolated spores or mycelium 
 to a sterilizing solution for a given period will kill them it is not safe 
 to conclude that a like exposure will kill them when they are in 
 contact with, but not embedded in, the tissues which at least in some 
 cases they may enter later. Had not previous examination shown that 
 such was not the case one might have concluded that the reason for 
 the growth of Alternaria in connection with some of the pieces of peel 
 or twig was because the mycelium had already penetrated the tissues 
 and was out of reach of the sterilizing solution. 
 
 STERILIZATION OF LEMON FRUITS AS A MEANS OF PREVENTING 
 
 ALTERNARIA ROT 
 
 The next step in the study was an attempt to at least minimize if 
 not entirely prevent this decay by sterilizing the fruits. The plan 
 followed was (1) to use mercuric chloride in the wash water in the 
 packing house, (2) to use "brogdite" 7 in the wash water in the 
 
 7 Brogdite is a commercial sterilizing medium that is being used in some 
 packing houses to prevent decay caused by blue and green molds. 
 
Bull. 408] ALTERNARIA rot of lemons 15 
 
 packing house, (3) to remove the buttons, and (4) to test the efficiency 
 of various sterilizing solutions in the laboratory, under controlled 
 conditions, provided the other methods did not prove to be effective 
 or practicable. 
 
 1. Tests were made in two packing houses. In one house five boxes 
 of silver lemons and twenty-two boxes of green lemons were washed 
 in each of the following: (a) water at approximately 95° F, (b) 
 mercuric chloride sterilizing solution at 60° F, and (c) the same 
 solution at 110° F. The lemons were retained in the solution approxi- 
 mately five minutes, then rinsed in the usual manner and put into 
 storage. 
 
 The fruit in the other packing house (three boxes of yellows and 
 five boxes each of silvers and greens) was treated in the same manner. 
 The fruit in both houses was examined at intervals for three and a 
 half months. 
 
 The results of the tests in these two packing houses indicate that 
 mercuric chloride can not be considered an efficient sterilizing medium 
 for the prevention of Alternaria rot in lemons. Apparently the cold 
 mercuric chloride solution had no controlling effect whatsoever, and 
 while the results with the hot solution indicated a slight degree of 
 control it was considered to be more of a retarding than an actual 
 sterilizing effect. 
 
 2. In the test with brogdite eight boxes of yellow lemons (1700 
 fruits) were divided into four lots of two boxes each. The first lot 
 was washed in water at approximately 113° F and the second, third, 
 and fourth lots were immersed in brogdite solution of the same tem- 
 perature for 5, 5, and 10 minutes respectively, after which they were 
 rinsed in cold water. The strength of the solution for the second lot 
 was sixteen ounces of brogdite to a gallon of water, and for the third 
 and fourth lots, twenty-four ounces. 
 
 By the end of two months 2, 4, 16, and 8 per cent respectively of 
 Alternaria rot had appeared in the four lots. AVhile only one test 
 was made with this solution at this time the results indicated that it 
 would be useless to repeat it. That more Alternaria rot appeared in 
 the treated than in the untreated lemons indicates that the treatment 
 produced or hastened some change in the lemon tissues which permitted 
 a more rapid invasion by the fungus. It should be said, however, 
 that there was no external indication of injury due to the treatment. 
 
 3. In each of four packing houses, six boxes of yellow lemons 8 and 
 six boxes of green lemons were given the usual treatment of washing 
 
 8 Although these lemons were yellow they were not classed as "tree-ripes. " 
 They had been prematurely colored by the winds and comparatively low tempera- 
 tures prevailing during November and December. 
 
16 
 
 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 and sorting. Three boxes of yellows and three of greens were placed 
 under storage conditions. The remaining three boxes each of yellows 
 and greens were sweated to such an extent as to cause at least most 
 of the buttons to drop. This was done to determine whether the 
 removal of the buttons would cause a reduction of the amount of Alter- 
 naria rot. In two of the houses the sweating was done with ethylene 
 gas and in the other two with oil stoves, The sweating periods in 
 packing houses 1 to 4 were 8, 8, 7, and 8 days respectively. The sweat- 
 ing caused no visible injury to the fruit. At the end of the sweating 
 period any buttons still attached were removed by hand. These boxes 
 of lemons were then placed in the storeroom beside the untreated 
 checks. All boxes of lemons were examined at intervals for four 
 months after the last boxes were placed in storage. The total number 
 of lemons in each lot showing Alternaria rot within that period is 
 shown in table 5. The exact number of lemons used was not deter- 
 mined but there were from 375 to 400 in each box. 
 
 TABLE 5 
 
 The Amount of Alternaria Rot in Lemons as Influenced by the Removal 
 of the Buttons. Three Boxes in Each Lot. 
 
 
 Sweated, buttons removed 
 
 Checks, buttons not removed 
 
 Packing house 
 
 Yellow 
 
 Green 
 
 Yellow 
 
 Green 
 
 
 Number of lemons showing Alternaria rot 
 
 1 
 
 157 
 55 
 20 
 46 
 
 1 
 
 13 
 
 3 
 
 49 
 
 223 
 30 
 21 
 
 118 
 
 2 
 
 2 
 
 3 
 
 3 
 
 2 
 
 4 
 
 2 
 
 
 
 The results indicate that the removal of the buttons from the 
 lemons is not a feasible method for the control of Alternaria rot. By 
 referring to the table it may be seen that in packing house No. 1 there 
 was apparently 30 per cent control and in house No. 4, 20 per cent, but 
 in house No. 3 there was no indication of control, and in house No. 2 
 there was 51 per cent more Alternaria rot in those lemons which had 
 the buttons removed than in the checks, 
 
 A similar test was later conducted in the laboratory. In this case 
 all of the lemons were silvers and the buttons were removed by hand 
 rather than by sweating. The fruit was brought directly from the 
 grove and stored in field boxes in a basement room of the laboratory 
 where the temperature did not vary more than four degrees, remaining 
 
BULL. 408] ALTERNARIA ROT OF LEMONS 17 
 
 for the entire time of the test (December 21 to March 5) at approxi- 
 mately 70° F. This temperature, although below the optimum, is very 
 favorable for the growth of Alternaria. The humidity of the room was 
 such that by the end of the test the lemons showed about the same 
 amount of shrinkage as would have occurred under the usual storage 
 conditions in the packing house. 
 
 On January 8 none of the fruits showed decay, but the tempera- 
 ture and humidity had been favorable for the germination of Alter- 
 naria spores that were attached to the buttons and to the surface of 
 the fruits. Small mats of mycelium could be distinguished very easily 
 with the unaided eye. The appearance of these mats of mycelium, 
 as they formed on the buttons in this and other tests and as they often 
 occur under packing house conditions, is shown in plate 2, figures A 
 and B. It was especially interesting that more mats of mycelium could 
 be seen in the button pits of those lemons from which the buttons had 
 been removed than could be seen in connection with the buttons that 
 remained attached to the lemons. This condition gives further evi- 
 dence that removal of the buttons does not necessarily remove all 
 chances of infection. Data to be presented farther on will show that, 
 although the button be removed, there are left in the crevices in the 
 tissues under the button a sufficient number of spores and bits of 
 resistant mycelium to cause infection as soon as conditions are 
 favorable. 
 
 By March 5, sixty -three cases of Alternaria rot had been found in 
 those lemons from which the buttons had been removed, and only six 
 in the checks, to which the buttons remained attached. These results 
 are comparable to those in packing house No. 2, as shown in table 5. It 
 may be of interest to note here that although the buttons in this test 
 were forcibly removed by using a clipper, such as is used in picking 
 lemons, only eight lemons in the entire lot showed a growth of 
 Penicillium. 
 
 Some of the reasons why removal of the buttons may favor infection 
 by Alternaria are (a) the removal of the buttons may weaken the 
 tissues, thus making them more susceptible to the attack of the fungus, 
 (b) the evaporation from the exposed tissues may make conditions 
 more favorable for the growth of the fungus, and (c) this excessive 
 evaporation may produce changes in the lemons which make them 
 more susceptible to attack. 
 
 Although only six lemons in the checks showed external signs of 
 Alternaria rot further examination disclosed the fact that a large 
 percentage of them were in the early stages of infection and would 
 soon have become decayed. This was determined by taking thirty 
 
18 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 lemons at random from among the checks. A thin slice was cut from 
 the stem end of each lemon in a manner such that the knife passed 
 just below the juncture of fruit and button, thus removing all crevices 
 which might be harboring spores or mycelium. A small disc of tissues 
 was then taken from each lemon immediately below the original point 
 of attachment of the button, and cultured to determine the number 
 that were infected. Twenty-one (70 per cent) of the thirty discs 
 showed an abundant growth of Alternaria. 
 
 The attempt to control Alternaria rot was continued by immersing 
 lemons in sterilizing solutions after the buttons had been removed. 
 In the first part of the test silver lemons that had just been picked 
 and washed were brought from three packing houses. The buttons 
 were removed from half of each lot and both lots were immersed for 
 five minutes in mercuric chloride of the usual strength, at 65° F. 
 They were then put into glass containers and placed on tables in the 
 laboratory where the temperature ranged from 54° to 72° F. Observa- 
 tions were made at intervals for a period of five months. The com- 
 parative amounts of Alternaria rot appearing in the different lots dur- 
 ing this time were : from house No. 1, 8 per cent in those without 
 buttons and 17 per cent in checks ; house No. 2, 67 per cent in those 
 without buttons and 68 per cent in checks; and from house No. 3, 
 8 per cent in those without buttons and 67 per cent in checks. 
 
 The second part of the test was like the first part except that only 
 one lot of lemons was used and the sterilizing solution was copper 
 sulphate (% °f 1 P er cent > as commonly used in lemon packing 
 houses). The results of this part of the test showed 51 per cent of 
 Alternaria rots in those lemons from which the buttons had been 
 removed and 57 per cent in the checks. 
 
 The results recorded in the two preceding paragraphs indicate that 
 it would be impracticable to attempt to control Alternaria rot in 
 lemons by sterilization, even after the buttons have been removed ; at 
 least this is true for the two sterilizing solutions used in this test. 
 
 ALTERNARIA IN THE LEMON TISSUE UNDER THE BUTTONS 
 
 The object of this test was to determine the percentage of fruit 
 infection after different periods of storage in different packing houses. 
 The lemons chosen were green or silver when picked. Some of them 
 were tested at once but most of them had been kept in storage for 
 from one to three months (see table 6). In order to reduce to a 
 minimum the possibility of error through contamination, the different 
 lots of lemons were immersed for five to ten minutes in mercuric 
 
Bull. 408] 
 
 ALTERNARIA ROT OF LEMONS 
 
 19 
 
 chloride solution at 115° to 120° F. After drying", the stem end of 
 each lemon was sliced off just below the juncture of button and fruit. 
 A cork borer was then inserted into the lemon so as to include the 
 tissues just below the point of attachment of the button. A second 
 cut was now made, parallel to the first, thus freeing a single disc of 
 tissue from each lemon, approximately % 6 inch thick and ^ inch in 
 diameter. All operations were performed in an inoculation cage and 
 with sterilized instruments. The discs of tissue, thirty from each lot of 
 lemons, were cultured on an artificial medium in Petri dishes and 
 incubated at 77° F. The percentages of discs from each lot of lemons 
 which showed infection with Alternaria are shown in table 6. 
 
 TABLE 6 
 
 Development of Alternaria from Discs of Tissue Taken from Beneath the 
 Buttons of Lemons that had just been Picked or had been Kept in 
 Storage. 
 
 Color of lemons when picked 
 
 Packing 
 house 
 
 Days lemons were 
 
 held in storage before 
 
 discs were cut 
 
 Percentage of discs 
 
 from which Alternaria 
 
 developed 
 
 Green 
 
 ( 3 
 
 1 
 
 2 
 
 4 
 < 
 
 3 
 
 2 
 
 4 
 
 1 
 
 56 
 61 
 61 
 64 
 84 
 92 
 93 
 99 
 
 3.3 
 6.7 
 6.7 
 0.0 
 
 
 10.0 
 0.0 
 0.0 
 3.3 
 
 Silver 
 
 6 
 
 7 
 
 8 
 
 10 
 
 11 
 
 10 
 
 I 
 
 9 
 8 
 11 
 3 
 4 
 5 
 
 2 
 
 2 
 
 2 
 
 2 
 
 2 
 22 
 40 ■ 
 49 
 50 
 55 
 55 
 59 
 60 
 75 
 
 0.0 
 0.0 
 0.0 
 0.0 
 0.0 
 0.0 
 33.3 
 
 
 0.0 
 3.3 
 0.0 
 
 50.0 
 0.0 
 6.7 
 
 70.0 
 
 The results of this test may be summarized as follows : 
 1. Of the 240 discs taken from lemons which were green when 
 picked, an average of only 3.8 per cent developed a growth of Alter- 
 
20 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 naria. Some of the lots that had been stored for three months showed 
 less decay than some of those stored only two months. 
 
 2. Of the 270 discs taken from the nine lots of stored lemons which 
 were silver when picked, an average of 18.1 per cent developed a 
 growth of Alternaria. 
 
 3. Of the 150 discs taken from the five lots of silver lemons two 
 days after picking none showed infection. 
 
 These data would indicate (a) that within certain limits (see 
 table 2) the higher the temperature the sooner the Alternaria fungus 
 will pass from the buttons into the lemon tissues and cause them to 
 decay, (b) that the more mature the fruit when picked the sooner it 
 will become decayed, (c) that the general vitality of the fruit is a very 
 important factor (e.g., see table 6, packing house 3, which shows 33.3 
 per cent infection in the silvers held 40 days and no infection in those 
 held 59 days), and (d) that the Alternaria fungus does not usually 
 pass from the buttons into the silver and green lemons until after 
 they have been picked and have begun to lose their vitality. 
 
 The relations mentioned in (d) of the preceding paragraph do not 
 hold true for lemons that have become tree-ripe before picking. 
 Several tests similar to the preceding one, have shown that in a com- 
 paratively large number of cases the fungus has passed from the 
 button into the tissues beneath by the time the tree-ripe lemons are 
 picked. This is especially true during the warmer months. 
 
 STERILIZATION OF LEMON BUTTONS 
 
 Buttons from mature lemons. — The tests already reported in this 
 paper have indicated that immersing the lemons, either with or with- 
 out the buttons, in mercuric chloride or copper sulphate solutions of 
 the strengths commonly used, does not control Alternaria rot. For 
 this reason an effort was made to find some method of sterilizing 
 detached buttons, since in this way much larger numbers could be 
 tested and the results much more quickly determined. It was hoped 
 that by this method some medium might be discovered which could 
 safely be used to sterilize the buttons while they were still attached to 
 the lemons. 
 
 The methods employed in this test are indicated in table 7, which 
 is a general summary of the results of many trials, with most of the 
 details omitted. Where HgCl 2 was used the strength of the solution 
 was always 1 to 1000. 
 
Bull. 408] 
 
 ALTERNARIA ROT OF LEMONS 
 
 21 
 
 
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22 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 The conclusions may be summarized as follows : 
 
 1. None of the solutions used can be considered to be efficient for 
 the prevention of the development of Alternaria in detached lemon 
 buttons. Higher concentrations, higher temperatures and longer 
 periods of exposure would doubtless effect greater degrees of steriliza- 
 tion of the detached buttons, but these would have to be so great that 
 the treatment would be impracticable under packing-house conditions 
 and, furthermore, would result in injury to the fruit. 
 
 2. Some of the solutions appeared to have more of a retarding 
 effect on Alternaria than on some of the other organisms such as 
 Mucor, Penicillium, Aspergillus, Colletotrichum and bacteria, and as 
 a result the largest percentage of infection is shown in the column 
 headed "Miscellaneous" (see lots number 12 and 21). That this effect 
 was due to retardation rather than killing of the Alternaria is at least 
 indicated by the results from lots 1 and 2 in which there was a marked 
 reduction in the percentage of Alternaria but no sterilization in 
 lot 1 and only 7 per cent in lot 2. Similarly, in a preceding test hot 
 water alone was used on pure cultures of Alternaria and the treat- 
 ment failed to kill the fungus but produced a very noticeable retarda- 
 tion in its development. From these results it appears that the 
 Alternaria in such cases as lots 1, 2, 12 and 21 was not actually killed 
 but that its rate of growth was temporarily inhibited or retarded to 
 such an extent that it was overgrown and further retarded by some 
 of the other organisms, and could not be detected when the cultures 
 were examined. Repeated tests have shown that hot water is not a 
 good sterilizing medium for the control of Alternaria rot, since its 
 retarding effect is apparently only temporary. 
 
 3. An examination of the data pertaining to the checks shows that 
 a very large percentage of the buttons was infected with Alternaria. 
 It seems very probable that many of those listed in the miscellaneous 
 column were infected with Alternaria but that it was outgrown by 
 other organisms. In lot 5, for example, 44 per cent of the buttons are 
 listed in the miscellaneous column. These buttons were taken from 
 lemons which, while practically mature, were on the trees during the 
 freeze of 1923. These particular lemons, when picked a few weeks 
 later, showed very slight, if any, visible injury, but it is of interest to 
 note that in the checks, 44 per cent of the buttons from these lemons 
 showed a predominant growth of Colletotrichum. Apparently the low 
 temperature had produced changes in the tissues that made them more 
 favorable for the growth of Colletotrichum than for that of Alter- 
 naria. This possibility is also proved by the fact that much more 
 Colletotrichum rot is found in lemons following a freeze than is found 
 
Bull. 408] 
 
 ALTERNARIA ROT OP LEMONS 
 
 23 
 
 under the usual conditions. With further reference to lot 5, it would 
 seem that mercuric chloride, as here used, is a better sterilizing agent 
 for Colletotrichum than for Alternaria, 
 
 The buttons from green and silver lemons are not reported 
 separately in table 7 since it appears that the vitality of the tissues 
 rather than their age is the principal factor determining the per- 
 centage of infection. Apparently the vitality of the tissues governs 
 the depth of penetration of the Alternaria and in turn this factor at 
 least partially determines the percentage of sterilization. 
 
 Buttons from young lemons. — In February buttons were taken 
 from immature lemons, of the average diameters shown in table 8. 
 Before the buttons were placed in the culture dishes they were soaked 
 in mercuric chloride solution of the usual strength for two minutes 
 at 65° F. 
 
 TABLE 8 
 
 Percentages of Alternaria, or Other Infection, Found in Buttons Taken 
 from Young Lemons of Different Sizes. 
 
 Number of buttons 
 
 Average diameter 
 of lemons 
 
 Alternaria 
 infection 
 
 Miscellaneous 
 infection 
 
 Sterile 
 
 50 
 50 
 50 
 
 Inches 
 
 l 12 /l6 
 
 lVl6 
 
 Vl6 
 
 Per cent 
 
 80 
 84 
 70 
 
 Per cent 
 
 8 
 
 6 
 
 24 
 
 Per cent 
 
 12 
 
 10 
 
 6 
 
 The data in table 8 indicate that a very high percentage of the 
 buttons, even from lemons that were only % 6 of an inch in diameter, 
 were at least potentially infected with Alternaria. It should be borne 
 in mind, however, that these buttons had been immersed in the 
 sterilizing solution for only two minutes and that at least not all 
 spores adhering to the surface, to say nothing of those which may be 
 protected in crevices, are killed by such an exposure. 
 
 In another test the larger crevices were eliminated by discarding 
 the enlarged central portion of the button, using only the compara- 
 tively thin outer layer of tissues which compose the calyx ring. The 
 sterilizing conditions were also made more rigorous. The calyx rings 
 were first soaked for two minutes in 95 per cent alcohol, to ensure a 
 complete wetting of the surface, and then for five minutes in 1 part 
 mercuric chloride to 1000 parts of 50 per cent alcohol. In this test the 
 calyx rings from lemons of two sizes were used. The larger lemons 
 were approximately l 1 /^ inches in diameter while the smaller ones were 
 picked just after the petals had fallen and were approximately % inch 
 in diameter. 
 
24 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 The results of this test showed 90 per cent Alternaria infection in 
 the calyx rings from the larger lemons and none in those from the 
 smaller ones. In the case of the older calyx rings this seems to indi- 
 cate either that the sterilizing agent did not kill all surface spores or 
 that the fungus had entered the tissues sufficiently far to be out of 
 reach of the sterilizing solution as applied. The results of a micro- 
 scopical study which will be reported later in this paper, indicate that 
 the latter explanation is the more probable. This conclusion is also 
 confirmed by the fact that the controls for the smaller sizes showed 
 60 per cent Alternaria growth while those which had been immersed 
 in the sterilizing solution showed none. This result indicates that the 
 treatment was sufficient to kill surface-adhering spores. Some por- 
 tions of the calyx rings from some of the smaller lemons had begun 
 to turn brown as if infection had started, but if this was the case the 
 penetration of the mycelium was so nearly superficial that its further 
 development was prevented by the sterilization process. 
 
 The high percentage of negative results obtained in attempting to 
 sterilize detached lemon buttons seem to indicate that attempted 
 sterilization of attached buttons, without injury to the fruit, is 
 impracticable. 
 
 SPRAYING TO CONTROL ALTERNARIA ROT 
 
 Experiments were conducted with a view to the control of Alter- 
 naria rot by means of orchard spraying. In one grove twelve rows of 
 twenty trees each were sprayed with Bordeaux (3-3-50) in April 
 just following the heaviest spring set of fruits. Six of these rows 
 were sprayed again in September following the heaviest fall set. In 
 a second grove 147 trees were sprayed in April, 72 of which were 
 sprayed again in November. 
 
 Cultures of buttons taken from the sprayed trees failed to reveal 
 any beneficial effect from the treatment. 
 
 During the course of the preceding spraying test a second and 
 more carefully conducted one was made in a different grove. Seven 
 hundred twenty lemon blossoms that had just opened and 500 young 
 lemons about % inch in diameter, were individually sprayed with 
 Bordeaux mixture (4-4-50) by a hand atomizer. Care was taken to 
 see that all parts of the blossoms, the small fruits, the calyx cups and 
 the adjacent p?rts of the stems were thoroughly wet by the spray 
 mixture. Seventy -seven days later the sprayed lemons, and controls 
 of a similar size, were picked and the buttons removed and cultured 
 to determine the relative percentages of Alternaria infection in the 
 
BULL. 408] ALTERNARIA ROT OP LEMONS 25 
 
 three lots. When picked, the lemons from the sprayed blossoms had 
 attained an average diameter of 2 % 2 inch while the fruits which were 
 !/4 inch in diameter when sprayed had become 1% 2 inches in diameter. 
 Buttons were taken from only one lot of controls and their average 
 diameter was approximately that of the larger of the two sprayed lots. 
 The percentages of infection appearing in the three lots of buttons 
 were (a) in those taken from the fruits from the sprayed blossoms, 
 57 per cent Alternaria, 43 per cent miscellaneous infection and none 
 sterile, ( b ) in those from the fruits y± inch in diameter when sprayed, 
 70 per cent Alternaria, 30 per cent miscellaneous and none sterile, and 
 (c) in the controls, 68 per cent Alternaria, 32 per cent miscellaneous 
 and none sterile. 
 
 The results of the two preceding tests, coupled with the fact that 
 there is an almost continuous setting of young fruits on lemon trees, 
 would indicate that there is little hope of satisfactorily controlling or 
 even materially reducing the percentage of Alternaria rot in lemons 
 by means of orchard spraying. 
 
 MICROSCOPICAL STUDY OF THE LEMON FRUIT AND BUTTON 
 TISSUES INFECTED WITH ALTERNARIA 
 
 The results of the tests reported thus far in this paper show the 
 comparative inefficiency of the usual methods as means of controlling 
 Alternaria rot of lemons. The inefficiency of these methods suggests 
 that spores or resistant mycelium were protected from the sterilizing 
 solutions by being in depressions or crevices in the button or in the 
 fruit beneath it, or that the mycelium had already penetrated the 
 tissues of the button or lemon and was out of reach of the sterilizing 
 solutions. To determine whether such could be the case, microscopical 
 studies were made of the tissues in question. For this purpose lemon 
 buttons and portions of the lemon tissues just beneath the buttons 
 were collected, killed, fixed, embedded, sectioned, stained with differ- 
 ential stains, and the sections studied microscopically to detect the 
 presence or absence of spores or mycelium on or in these tissues. 
 
 Weak chrom-acetic acid seemed to give better results than any of 
 the several other killing solutions tried. Of the several different stain 
 combinations commonly used for differentiating fungus mycelium and 
 host tissues, such as Pianeze, Delaneld's Haematoxylin and Eosin, and 
 Mazdala Red and Light Green, the last gave the best results. 
 
 The age of the lemons from which the buttons and other tissues 
 were taken is indicated by the fact that all of them had been picked 
 previously for commercial purposes. The color of the lemons at time 
 
26 
 
 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 of picking and their treatment during the interval between picking 
 and the time at which the buttons and lemon tissues were selected for 
 this study is stated in the legends of plates 2 and 3. 9 Figures 1 to 12, 
 plates 2 and 3, are photomicrographs of portions of some of the sec- 
 tions of the stained tissues. The magnifications were the same for all 
 of these figures. 
 
 Fig. 1. — Outline of a projection of a microscopical section which shows the 
 configuration of a button and its attachment to the fruit. The numerals placed 
 on different portions of the outline designate the approximate regions of the 
 buttons or fruits from which the sections were taken which are figured in 
 plates 2 and 3. 
 
 The microscopical study of the tissues in question indicates : 
 
 1. The ineffectiveness of sterilizing agents seems to be due to the 
 presence of spores and mycelium in depressions or crevices where they 
 are inaccessible to the sterilizing solution, and to penetration of 
 mycelium into the button tissues where it is out of reach of the 
 sterilizing solution (pi. 2, fig. 4, and pi. 3, figs. 5, 6, 7, and 8). 
 
 2. In some cases spores and resistant mycelium were found attached 
 to the surface of the button but with no evident attempt at penetration 
 (pi. 2, fig. 2). In other cases apparently the spore had germinated 
 on the surface and had made an unsuccessful attempt at penetration, 
 but had produced certain changes in the button tissue directly beneath 
 (pi. 2, fig. 1). In most cases where the fungus mycelium had gained 
 entrance the tissue of the button had formed a, resistant or semi- 
 resistant layer of tissue in advance of the invasion of the fungus 
 (pi. 2, figs. 1, 3, and 4, and pi. 3, figs. 6 and 7). The cells composing 
 the resistant layer are or at least very closely resemble young cork 
 cells. They are almost or entirely free from protoplasmic contents 
 and their exterior walls are thicker than their lateral or inner walls. 
 
 9 To visualize the region of the buttons or lemons from which figures 1 to 12, 
 plates 2 and 3, were taken, frequent reference should be made to text figure 1. 
 
BULL. 408] ALTERNARIA ROT OF LEMONS 27 
 
 A cork cambium is very evident in most cases and some of the pre- 
 existing cells which were pushed off by the newly-formed cork layers 
 are found intermingled with the mats of spores and mycelium on the 
 surface (pi. 2, figs. 3, and 4, and pi. 3, fig. 7). In some cases the 
 fungus seems to have been able to pass this barrier imposed by the 
 host, and as many as three successive layers of cork tissue have been 
 formed at intervals in the effort to check the invasion of the fungus. 
 
 3. In some cases the invasion of the fungus was so rapid that 
 apparently the host tissues did not have time to form the resistant 
 layer of cork cells until considerable progress had been made by the 
 fungus. Plate 3, figure 5 shows such a case which occurred under 
 field conditions (compare pi. 2, figs. 1 and 3, and pi. 3, figs. 6 and 7). 
 Under field conditions the vitality of the tissues of the button and 
 lemon fruit is usually at its maximum while conditions for the 
 germination and growth of the fungus are usually unfavorable. But 
 when the lemons have been picked and stored the conditions are 
 reversed and the fungus makes much more rapid progress. The result 
 of an exaggerated case of this reversal of conditions is shown in plate 3, 
 figures 9 and 12. In this case buttons from green lemons were placed 
 in Petri dishes containing an artificial medium very favorable for the 
 growth of the fungus. Had these lemons, with their buttons attached, 
 been placed under usual storage conditions it probably would have been 
 at least three months before the fungus would have progressed through 
 the buttons and into the lemons to such an extent as to cause visible 
 signs of decay. Under the favorable conditions in the Petri dish the 
 fungus penetrated all portions of the buttons in eight days and so 
 modified their internal structure that the individual cells had lost 
 their identity, except in the case of the more resistant wood cells. In 
 the prepared sections the interior of the buttons appeared to be almost 
 a homogeneous mass with the mycelium penetrating all portions of it 
 (pi. 3, fig. 12). The figure just mentioned should be compared with 
 figure 11 in the same plate, noting comparative diameters of the 
 mycelial threads and the nature of the host tissues. Figure 11 repre- 
 sents the conditions in a button that had been taken from a lemon 
 kept in the sweatroom for ten days and then held in storage for 
 another ten days. 
 
 4. As a rule the fungus mycelium has not passed from the button 
 down into the fruit tissues at the time of picking. Exceptions to this 
 rule may be found in cases where, the lemons have become over-ripe 
 before picking or where they have been exposed to excessively high or 
 low temperatures. Apparently the penetration does not take place 
 until the lemon tissues have become "weakened" (changed physio- 
 
28 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 logically) to a certain extent. It seems also that any environment 
 which produces a weakened condition in the fruit, such as a com- 
 paratively high storage temperature, also produces conditions favor- 
 able for rapid growth of the fungus. The combination of these two 
 effects results in a more or less rapid decay of the fruit. 
 
 5. The microscopical study further showed why very many of the 
 stored lemons may be subject to the attack of Alternaria even though 
 the buttons have been removed and the lemons soaked in a sterilizing 
 solution before being placed in storage. Those who are familiar with 
 the early stages of development of the lemon fruit know that after 
 the petals have fallen there is left behind the calyx cup, in the center 
 of which the young fruit is attached. This calyx cup makes a suitable 
 receptacle for catching Alternaria spores. As the fruit enlarges, the 
 edges of the calyx cup come in contact with the fruit and the Alter- 
 naria spores are thus entrapped. In this manner some of the spores, or 
 bits of resistant mycelium from spores that germinated and grew for 
 only a short time, may easily find lodgment in some of the minute 
 depressions or crevices in the fruit under the button. Again, as the 
 fruit enlarges it no longer maintains an upright position but hangs 
 downward and in many cases portions of the edges of the button lose 
 their close contact with the fruit. This condition affords another 
 chance for the entrance of spores into the space between the button 
 and fruit. These spores or resistant bits of mycelium are not reached 
 by the sterilizing solutions under the usual methods of application. 
 Plate 3, figure 8 shows two small pieces of resistant mycelium in a 
 microscopical crevice in the surface of a lemon just beneath the 
 button. 
 
 6. In describing the characteristic appearance of the lemon tissues 
 beneath the button, in the early stages of infection with Alternaria, it 
 was stated in a previous section of this paper that the individual 
 vascular bundles appeared to be infected first, as indicated by the fact 
 that they are first to show discoloration. The microscopical study has 
 shown that initial infection probably takes place first in the phloem, 
 or in the bundle sheath, and passes from there into the surrounding 
 parenchyma or into and among the water-conducting vessels and other 
 xylem elements. Plate 3, figure 10 shows mycelium among the wood 
 cells and medullary rays and in a water conducting vessel. No evi- 
 dence was found to indicate that the Alternaria mycelium enters the 
 water-conducting vessels of the button, while the lemon is still attached 
 to the tree, to such an extent as to restrict the passage of water. 
 
BULL. 408] ALTERNARIA ROT OF LEMONS 29 
 
 DISCUSSION OF CONTROL MEASURES 
 
 The commonly used methods of spraying and sterilization appear 
 to be in a large measure ineffective as a means of controlling Alter- 
 naria rot of lemons. The ineffectiveness in the case of spraying is very 
 largely due to the fact that new fruits are being formed almost con- 
 tinuously and that Alternaria spores are always present in the air 
 and on all parts of the tree. These spores are constantly being carried 
 to the newly formed tissues either by air currents or rain. The 
 difficulty in sterilizing the lemon fruits after they have been picked 
 lies in the fact that the mycelium has already entered the button tissues 
 or that the spores are located in crevices in the button or in the lemon 
 tissue beneath the button, so that both mycelium and spores are 
 inaccessible to the sterilizing solutions commonly used for such pur- 
 poses. Various new solutions are being tested by different workers 
 and it is hoped that one may be found which- will be able to effect 
 sterilization without injury to the fruit. 
 
 Rogers and Earle, 10 who worked at San Pedro, Isle of Pines, on 
 control measures for stem-end rot 11 of several types of citrus fruits, 
 recommend that the fruits be pulled instead of clipped from the tree 
 and that the scar on the fruit, left by detaching it from the stem, be 
 covered with shellac. An initial test of this method proved it to be 
 so inefficient for the control of Alternaria rot that it was not given 
 further attention. Even were this method more or less effective it does 
 not seem that it would be practicable under California conditions 
 where such a large crop of lemons is produced. Winston, Fulton and 
 Bowman, 12 working in Florida, have found that both orchard spraying 
 and the removal of the buttons from the fruits very noticeably reduce 
 the amount of stem-end rot in oranges and grapefruit. They have 
 found also that pruning the dead branches out of the trees tends to 
 reduce stem-end rot, especially where Diplodia is concerned. The 
 almost universal presence of Alternaria spores on decaying vegetation, 
 coupled with the fact that the new fruits are forming almost con- 
 tinuously, indicates that pruning would be comparatively ineffective 
 in controlling Alternaria rot of lemons. 
 
 10 Bogers, J. M., and F. S. Earle. A simple and effective method of protect- 
 ing citrus fruits against stem-end rot. Phytopathology 7: 361-367. 1917. 
 
 11 Stem-end rot is somewhat similar to Alternaria rot in that the fungus 
 enters the fruit through the stem end. It is principally caused by Phomopsis 
 citri and Diplodia natalensis. 
 
 12 Winston, J. E., H. E. Fulton, and J. J. Bowman. Commercial control of 
 stem-end rot. The Florida Grower 28: 6, 26. 1923. 
 
30 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 
 
 With reference to the removal of the buttons as a means of control, 
 it ordinarily requires a week or more to sweat green lemons to give 
 them the desired color. Under such conditions the buttons remain 
 attached to the lemons. If the conditions were intensified enough 
 during this process to cause the buttons to drop there would be the 
 added danger of injury to the fruit and also the probability of causing 
 physiological changes in it which would favor the growth of Alternaria. 
 In Florida it is stated that an exposure to ethylene gas, at a tem- 
 perature of 80° to 85° F and a humidity of 85 to 90 per cent for 
 thirty-six hours will cause orange and grapefruit buttons to loosen. 
 
 At the present time and under California conditions it is not 
 possible to prevent Alternaria rot of lemons satisfactorily, but it has 
 been found that the losses from this disease may be materially reduced 
 by the adoption of the following suggestions : 
 
 1. The trees should be kept as nearly as possible in a healthy condi- 
 tion so that the fruit will grow continuously from setting to maturity. 
 Fruits that have experienced a set-back, due to lack of water or other 
 cause, are weaker than if they had grown continuously and hence are 
 more susceptible to attack by Alternaria, 
 
 2. The fruit should be picked while it has a high vitality, i.e., 
 while it is yet silver or green in color. The Alternaria rot fungus 
 cannot attack it as long as its vitality is high. 
 
 3. Washing the fruit in hot water materially raises its temperature. 
 If this temperature could be reduced before the fruit is placed in 
 storage the chances for the development of Alternaria rot would 
 probably be reduced. It is a well-known fact that a sudden reduction 
 of temperature, following a comparatively high one, markedly retards 
 the development of various diseases, 
 
 4. If the fruit can be dried sufficiently to remove all free water 
 from beneath the buttons before the fruit is placed in the storeroom, 
 conditions are made less favorable for the development of Alternaria. 
 
 5. Minimum temperature, humidity and time of exposure should 
 be used when sweating green fruit. 
 
 6. So far as possible, all fruits affected with endoxerosis (internal 
 decline, blossom-end decay, etc.) should be excluded. 
 
 7. The fruit should be stored at a low temperature. The develop- 
 ment of Alternaria rot in lemons is extremely slow at a temperature of 
 55° F, or lower, unless the fruit has a low vitality. 
 
 8. During the process of grading and packing, an unnecessarily 
 long exposure of the fruit to the warmer temperature of the grading 
 room should be avoided. 
 
BULL. 408] ALTERNARIA ROT OP LEMONS 31 
 
 9. Before packing for shipment each lot of fruit should be care- 
 fully inspected for the presence of Alternaria rot in its early stages, 
 i.e., before it is visible on the surface. This inspection should be made 
 by slicing off the stem end of the lemon, just under the button. If 
 the lemon has become infected the small bundles of tissues in the 
 center of the cut surface will have a pinkish to light brown color. If 
 a given lot of fruit examined in this manner shows more than an 
 extremely low percentage of infection it should not be shipped, or if 
 shipped it should be placed on a market where the time consumed in 
 transit will be short and where the chances for its early consumption 
 are good. 
 
 SUMMARY 
 
 1. Alternaria probably causes more California lemon fruits to 
 decay than any other one fungus, with the possible exception of 
 Penicillium. 
 
 2. Alternaria rot may be found in all lemon-growing districts of 
 California. 
 
 3. The lemon fruits become potentially infected with Alternaria 
 before picking, but they do not usually begin to decay until after 
 being placed in storage. 
 
 4. Alternaria rot of lemons is caused by the fungus Alternaria; 
 endoxerosis of lemons is not caused by a fungus but by abnormal 
 physiological conditions. Care should be exercised not to confuse 
 the two. 
 
 5. The temperature range most suitable for the maximum growth 
 rate of Alternaria either in the lemon tissues or in the culture, is 
 approximately 78° to 83° F. 
 
 6. Isolated Alternaria spores or resistant mycelium may be killed 
 by a two-minute exposure to 1 :1000 mercuric chloride, but if they 
 are in contact with the surface of the lemons, buttons, or twigs, it 
 appears that an exposure of at least six to eight minutes is required 
 in order to kill them. 
 
 7. To attempt to prevent Alternaria rot of lemons by the methods 
 commonly used for such purposes seems impracticable. 
 
 8. The removal of the buttons before placing the lemons in storage 
 is not effective in controlling Alternaria rot, and, furthermore, such 
 a procedure would be impracticable. 
 
 9. Although the button becomes infected while the fruit is very 
 young and spores are also entrapped under the button, it is only in 
 exceptional cases that the Alternaria fungus has entered the fruit 
 tissues by the time the lemons are picked. 
 
32 UNIVERSITY OP CALIFORNIA EXPERIMENT STATION 
 
 10. Alternaria may enter the lemon directly from the button or 
 from spores or mycelium harbored between the button and the lemon. 
 
 11. If lemons of average vitality are stored at a temperature of 
 55° to 60° F, Alternaria rot will usually begin to appear in tree-ripes 
 in from one month to six weeks, in silvers in from two to two and a 
 half months, and in greens from three to four months. 
 
 12. Present methods of orchard spraying appear to be both unsuc- 
 cessful and impracticable for the prevention of Alternaria rot of 
 lemons. 
 
 13. A microscopical study of the button and lemon tissues con- 
 cerned has shown (a) attempts at sterilization were unsuccessful 
 because spores and resistant mycelium were located in depressions or 
 crevices where they were inaccessible to the sterilizing solution and 
 because in most cases some of the mycelium had already penetrated 
 the button tissue where it too was out of reach of the sterilizing solu- 
 tion, (&) the button tissues retard the advance of the fungus, after 
 it has once entered, by the formation of a corky layer, (c) as the 
 Alternaria enters the fruit it first follows the bundle sheath and then 
 it passes into the parenchyma and into the water-conducting vessels 
 and other wood elements, and (d) crevices and depressions in the 
 lemon tissues under the button harbor spores and resistant bits of 
 mycelium which, though the button be removed, may produce infection 
 as soon as conditions become favorable. 
 
 14. Certain methods of picking, storing, etc., will materially reduce 
 the amount of loss due to Alternaria rot. 
 
 It may be said in conclusion that while Alternaria is the organism 
 which directly produces the decay under discussion in this paper, it 
 is realized that some of the other organisms, often found associated 
 with the Alternaria, may contribute toward making conditions more 
 favorable for its development. 
 
 ACKNOWLEDGMENTS 
 
 The author wishes to acknowledge the helpful suggestions given by 
 Dr. H. S. Fawcett, the assistance given by Mr. C. 0. Smith in making 
 and examining many of the cultures, and the hearty cooperation of 
 many of the Lemon Associations during the course of this investigation. 
 
PLATE 1 
 
 In comparing these figures illustrating the characteristics of endoxerosis 
 and Alternaria rot of lemons, note especially the initial points of attack, the 
 color differences, and the conditions of the affected tissues. 
 
 Fig. 1. A green, but practically mature, lemon showing an early stage of 
 endoxerosis. Note deposits of gum near stylar end of lemon and in the peel 
 at one side, and the more or less typical precoloring of the surface of the peel 
 at the stylar end. 
 
 Figs. 2 and 3. Two different types of development in the later stages of 
 endoxerosis. 
 
 Fig. 4. A comparatively early stage in the development of Alternaria rot. 
 Note area of initial infection under the button, also the advancing area of 
 infection down the center of the lemon. 
 
 Fig. 5. Intermediate stage in the development of Alternaria rot. Even at 
 this stage it is not possible to detect the infection without cutting the lemon. 
 
 Fig. 6. A later stage of Alternaria rot. Following this stage the entire 
 fruit becomes infected and becomes a dark-brown to almost black, soft mass 
 of decaying tissues. 
 
 [34] 
 

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 -i 
 
 
 \ 
 
 / 
 
 2 
 
 3 
 
PLATE 2 
 
 To visualize the region of the button or fruit from which figures 1 to 12, 
 plates 2 and 3, were taken, reference should be made to text figure 1. 
 
 Figs. A and B. Mats of Alternaria mycelium growing from the buttons of 
 lemons that had been stored where the temperature and humidity were too high. 
 
 Fig. 1. From the button of a silver lemon just after picking. Resistant 
 spores and hyphae, below which cork cells have formed. 
 
 Fig. 2. From the button of a green lemon just after picking. Masses of 
 resistant spores adhering to the surface of the button. The tissues below 
 apparently unaffected. 
 
 Fig. 3. From the button of a silver lemon just after picking. Masses of 
 resistant spores above the infected tissue which is penetrated by hyphae. Note 
 resistant layer of cork cells below infected tissues. 
 
 Fig. 4. Portion of a crevice in a button from a green lemon that had been 
 in storage ten days. During this period the temperature ranged from 75° to 
 85° F and the air was almost saturated with moisture. Note that there are 
 hyphae in upper half of figure but none below the resistant corky layer in the 
 lower half of figure. 
 
 [36] 
 
; ^ 
 
 B 
 
 ...X 
 
PLATE 3 
 
 Fig. 5. From the button of a yellow lemon just after picking. Clumps of 
 resistant hyphae formed on the surface of the infected tissues below. 
 
 Fig. 6. Source, same as for figure 5. Eesistant hyphae in the infected 
 tissues. Note again corky resistant layer below. 
 
 Fig. 7. Source, same as for figure 5. Portion of a crevice containing spores 
 and hyphae; their advance at least retarded by the corky resistant layer. 
 
 Fig. 8. From a green lemon sweated ten days and then held in storage 
 ten days longer. Enlarged ends of hyphae in one of the microscopical depres- 
 sions or crevices which appear in the surface of the lemon under the button. 
 
 Fig. 9. From button of a green lemon just picked; button removed, placed 
 in semesan sterilizing solution in 90 per cent vacuum for one hour, then plated 
 on glucose-potato agar for eight days. Note vigorous growth of fungus in the 
 tissues under these conditions. 
 
 Fig. 10. Source, same as for figure 8. Hyphae showing in a tangential 
 section of the wood tissue of a button from a green lemon. 
 
 Fig. 11. Source, same as for figure 8. Hyphae in parenchyma tissue of 
 lemon; about one-quarter inch below button. 
 
 Fig. 12. Source, same as for figure 9. Hyphae in cortex parenchyma of the 
 lemon button. 
 
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