J. F. L. CHILDS AND E. A. SIEGLER
U. S. D e p a r t m e n t o f A g r ic u ltu r e , O rlan d o, F la.
P
R E L IM IN A R Y reports have been made recently on the effectiveness of thiourea (2) and several other organic compounds (3) in controlling.decays of Florida orange fruits. Tests with thiourea over two seasons and additional tests with thio
acetamide, 2-aminothiazole, and quinosol (8-hydroxyquinoline sulfate) confirm the results previously reported. Although the investigations are still in progress, the problem o f decay control in citrus fruits is so important economically that it is desirable to report at this time on the status of the work.
T he first comprehensive publication on control o f orange decay appeared in 1908 (17). Since then citrus fruits have taken first place in economic value, and improved methods o f handling and increased use o f refrigeration in transit have decreased the loss from decay on a per box basis; nevertheless the total loss has increased with the tremendous increase in production. Re- frigeration in transit, however essential for the delivery of fruit in a fresh condition to the wholesale markets, merely delays the incidence of decay and transfers the main loss to the retailer, the consumer, and ultimately back to the producer. For this reason there have been many attempts to develop a treatment which would decrease the spoilage of fruit after its arrival at the whole
sale market.
In this problem most of the critical etiological factors have been known for many years. The stem-end rots are caused by the fungi D iplodia natalensis and Phom opsis c it r i; the blue and green molds, respectively, are caused by the fungi P en icilliu m
■ilalicum and P . digitatum. All of these organisms are dissemi
nated by spores. The stem-end rot organisms infect the “ button”
(receptacle, calyx, and stem parts) o f the fruits some time before value; others have not justified the expense involved. D ecay of Florida grapefruit caused by D iplodia and Phom opsis has been is pulled, there is often increased loss from green mold.
Despite the degree o f control secured by remedial measures applied in the grove, the need for a method which will practically ensure 100% control is apparent when the problem is viewed from the standpoint o f the various groups that comprise the citrus industry. Each group recognizes that intangible liabilities are in
herent in fruit 'which arrives at the consumer market with latent, invisible infection; but since the loss by each group is only a fraction of the total, there has been little appreciation of the enormity of the aggregate loss. In experiments at this laboratory over two seasons it was found that oranges stored three weeks at 70° F. with about 70 to 80 % relative humidity showed 20 to 60%
decay. The major portion o f such losses is borne by the consumer.
One of the earliest control measures used commercially was the borax dip, developed about 1923 (8). This has remained the standard for comparative tests with hundreds of other antiseptics and fungicides. T o be m ost effective under Florida conditions, it is essential that borax be applied to the fruit shortly after harvest
January, 1946 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y 83
and before exposure to ethylene gas (1). Since that procedure is not always convenient, control suffered and the method fell into disuse. A proprietary compound known as D owicide A (sodium o-phenyl phenate) is used in some packing houses. The same ma
terial is also used {15) in conjunction with formaldehyde (to de
crease rind injury).
In 1935 Tompkins reported {21) that paper wraps impregnated with diphenyl were effective. The vapors from this material are fungistatic in that they arrest the growth of the organisms caus
ing decay {1 8 ,2 1 ).
The effectiveness of thiourea in decay control was brought out during a search fo r a control method that could be applied in the grove. A material was sought which would prevent further growth o f the latent D iplodia amd Phom opsis infections or would cause abscission o f the buttons after the fruit was picked. T o this latter objective, many synthetic plant hormones and other physiologically active compounds were tested. F. D . Jones, of American Chemical Paint Company, suggested that benzothiazyl- thiazoglycolic acid and thiourea were worth a trial. In field tests thiourea sprayed on the trees failed to cause abscission of the buttons and resulted in foliage injury. In laboratory tests good decay control was. obtained by treating the fruit after har
vest. Examination of the literature revealed that thiourea de
creased damping-off of cuttings in several susceptible plants, when applied with root-initiating hormones {6 ). I t has been reported toxic in vitro to several species of the dermatophytes, Trichophyton and A chorion {10), and moderately active in the chemotherapy of pathogenic and saprophytic mycoses o f animals {13). Its presence is reported in several species of fungi {12) and in one species o f angiosperm {16).
Preliminary results with thiourea led to a search for other com
pounds of equal control value; three other materials, quinosol, thioacetamide, and 2-aminothiazole have given encouraging re
sults. Though seldom used in agriculture, the fungicidal proper
ties o f quinosol are well known {1 9 ); it has been reported to con possibility o f deleterious effects when treated fruit is consumed.
In this report there is no intentional implication that any o f the four compounds that have given experimental control will suc
cessfully meet the public health requirements or other conditions necessary before a new treatment can be put into commercial practice. I t should be mentioned, however, that the remarkable results obtained with at least two of these materials may
cast the discovery o f others that will meet all requirements, if one o f these does not. Thus the fact that thiourea gave excellent con
trol was the opening wedge which led to tests with the other compounds. Of equal importance is the experimental evidence which offers a logical basis for the fungicidal activity of three of these compounds {8) and should serve as a helpful guide for future selections.
The tests have been sufficiently comprehensive to include the salient points considered pertinent to packing house operations in the event that any of these materials can m eet legal requirements and be justified on the basis of cost.
M E T H O D S
Oranges used in this work were usually clipped directly from the trees, but several commercially picked lots were obtained from packing houses. The varieties were those grown commercially in Florida such as Parson Brown, Pineapple, Seedling (sweet orange), and Valencia. In most cases the fruit was exposed to ethylene vapors for approximately 40 hours before treatment, primarily to predispose it to D iplodia stcm-end decay and also because this “ dcgreening” process is widely used in packing houses at certain seasons o f the year. After treatment the fruit was stored at 70° F. for 3 weeks, and at the end of that time the total number of decayed fruit was determined. The temperature of 70° F. approximates that at which the fruit would be held in the home and also is favorable for growth of all the decay organ
isms involved. N o attem pt was madb to control humidity, which normally averaged about 70 to 80% .
In the course o f this work seventy-one compounds in the following groups were tested: eight inorganic sulfur compounds, nine organic acids, six iodine compounds (organic and inorganic), fourteen plant-hormonelike compounds, fifteen organic sulfur compounds, fifteen proprietary organic fungicides or synthetics, and four unclassified organic compounds.
Unless otherwise specified, the immersion treatment in all ex
periments was a momentary dip of 5 seconds or less in water solu
tions of the several compounds. Vatsol OT, 0.05% , was used as wetting agent, except with quinosol with which it is incompatible.
Tests showed that the addition of a wetting agent did not im
prove decay control, but its use was continued for the sake of more uniform coverage and drain-off.
R E S U L T S
The concentrations o f thiourea which control stem-end rots and blue and green mold decays were investigated in a series of tests in which solutions of 0.1 to 10.0% were used. The data in Table I are the aggregate o f four to eight replications at each con-Sound
84 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y V ol. 38, No. 1 concentrations o f 4 % and above there was no material difference in effectiveness. A concentration of 1.0% shows roughly 5 0 % control. Similar experiments were made to determine the effec
tive concentrations o f thioacetamide and quinosol. The results with thioacetamide (Table I) indicate that it closely parallels thiourea in effectiveness. Quinosol is slightly less effective in the control o f both types of rots (Table I).
Inconsistent results characterized the tests with 2-aminothi- azole in that decay control fluctuated from poor to excellent. This compound is relatively insoluble (1.0% at 130° F. is turbid) but a series of tests were made with 5 .0 % concentrations at tempera
tures o f from 82° to 130° F. Above 130° rind injury is en high toxicity to several organisms and some showed toxicity to all four; but with the exception of the four compounds discussed in this paper, none showed any control of decay in dipping tests with orange fruits. In this connection it should be mentioned that urea and thioacetic acid, closely related to thiourea and to thioacetamide, respectively, showed no measurable decay control in several tests. Urea was less toxic than thiourea to all four organisms in vitro; thioacetic was much more toxic than thioacetamide. This emphasizes the fact that the criterion of toxicity in vitro, so frequently used in the large-scale testing of fungicides, may have little relation to effectiveness in controlling decay and other diseases.
E F F E C T O F T I M E O F IM M E R S IO N
The relation of the length of immersion to decay control was investigated by dipping lots of fifty oranges in a 1.0% solution of thiourea for (a) 5 seconds, (b) 1 hour, and (c) 4 hours. The ex
periment was repeated twice. The fruits were washed with water after dipping to remove any thiourea that had not penetrated. the controls averaged 31.6% decay.
Ta b l e I I I . Ef f e c t o f Im m e r s i o n Pe r i o di n 1 % Th i o u r e ao n thiourea-treated fruit for human consumption,' it was desired to determine what quantity, if any, penetrates into the juice, what quantity penetrates into the rind, and how much remains on the outside. It was found that fifty dry, clean oranges (26.3 pounds), dipped in a 0.05% solution o f Vatsol O T and drained, had removed 33 ml. o f solution. Fifty clean, wet oranges (26.0 pounds) removed only 14 ml. Hence when dry fruit is of orange juice rendered colorimetric determinations difficult;
but by making a series o f thiourea dilutions in the juice o f un
o f rind weight. When the time elapsed and the handling involved in making decay counts are considered, the discrepancy between the total thiourea found by analysis and that calculated on the basis o f solution removed from the dipping tank is not considered serious.
A D A P T A T IO N T O P R A C T IC E
In attempting to adapt thiourea and other compounds to com
mercial practice a number o f questions arose, such as choice of
acetamide and, to a lesser degree, with the other two compounds.
However, much of the early work and consequent elucidation of waxes. Thiourea, thioacetamide, and 2-aminothiazole are com
patible with several such waxes but quinosol is not. A series of
formed with thioacetamide and with 2-aminothiazole. Water- waxes without thiourea or other decay inhibitors showed no con
trol o f decay.
Quinosol, which is not compatible with the water-waxes tested, was used in conjunction with the solvent-waxing method in which the wax is dissolved in a volatile solvent such as benzene and applied as a mist-type spray. After momentary immersion in a 5 % solution of quinosol, the fruit was allowed to dry before ex
posure to the solvent-wax spray. Total decay in the treated lots was 6.2% , and 34.5% in the checks at the end of 3 weeks (Table V). Quinosol solutions do not wet the rind surface readily.
O f the six emulsifiers and wetting agents tested, only Tween 80 was compatible with quinosol and was used in these tests at 0.75
January, 1946 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y 8S shown no evidence of decay control in comparison with untreated fruit.
In commercial practice, however, fruit to be treated by the solvent-wax must first be dried and polished. This is customarily accomplished by passing the wet fruits over a long series of roller brushes while exposed to currents of warm air. T o determine whether such a drying procedure would decrease control of decay, a series o f experiments was made with fruit which was brush-dried after treatment and then sprayed with solvent-wax.
The results (Table V) show 6 .5 % decay in the treated fruits and 32.7% in the checks. When the fruits were rinsed before brush drying, there was a further decrease in control with 19.6% decay in the treated fruits and 26.1% in the check lots (Table V).
As will be discussed later, a slight modification of the treatment in the solvent-wax method should result in better control.
An important question is the length o f the period of protection afforded by these chemical treatments. Although m ost of the experiments were concluded at the end of 3 weeks, occasional lots of thiourea-treated fruit were stored at 38° and 50° F. for 2 two more fruits had rotted (with green m old), an increase o f only
Ta b l e V. Ef f e c t o f 5 % Co n c e n t r a t i o n o f Co m p o u n d.
0.8 % . The fruit was in reasonably good condition both in appear
ance and for consumption.
D IS C U S S IO N A N D C O N C L U S IO N S
Some of the factors that affect decay control should be reviewed to provide a perspective for evaluating the results. These factors are best considered in relation to their effect on the type and amount o f decay encountered. Although all four types of fungi are more abundant in the older groves, there is con siderable fluctuation in the prevalence o f individual fungus species. The interrelation o f grove site, season of harvest, maturity o f picked fruit, and effect of gassing with ethylene is shown by the various types and amounts of decay in different samples of fruit. In view of this wide variability, the uniformly high degree of control in the experiments reported here is surpris
ing. There was little tendency in these tests for the total decay of the treated lots to fluctuate as it did in the untreated lots.
Fortunately the total percentage of decay was remarkably
uni-86 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y V ol. 38, No. 1 form in randomized samples o f fruit from a single picking in a
given grove. Occasional fruit rot (less than 1 % of the total) caused by various other organisms was classified as side rots, but data are omitted from the tables for the sake o f brevity since their control parallels that of the other rots.
The experiments on the relative effectiveness o f thiourea, thiacetamide, and quinosol at different concentrations (Table I) provided the basis for the selection of concentrations for use in small-scale packing house tests. It is seen that 5 % is well within the range o f effective concentrations. A t this concentration a slight dilution in the course of an experiment, occasioned by dipping fruit still wet from a previous rinsing, should affect the results very little and at the same time permits a short immersion period. I t may prove feasible under commercial conditions, however, to decrease concentrations somewhat without detriment except for an occasional lot of heavily infected fruit. W ith lower concentrations it should be possible to regulate penetration and consequent amount of decay by varying length of immersion period as indicated in Table III. If tolerance limits for the chem
ical are established, this method might serve a useful purpose.
Penetration o f fruit tissues is definitely associated with the highly effective action of these compounds in controlling citrus decay. It was noted earlier that infection by the stem-end rot obtained commercially. In the borax treatment (I) satisfactory control is obtained only when the fruit is dipped soon after pick
ing. If treatment is delayed several days by necessary packing house handling, the degree o f control drops to a point o f question
able benefit.
The colorimetric analyses, in which approximately 12 p.p.m. of thiourea was found in the juice and 20 p.p.m. in the rind of treated oranges, constitute direct evidence that thiourea pene
trates into orange tissues. Supporting evidence is the fact that pro
longed immersion (1 hour) in 1.0% solution gave control whereas momentary dipping at this concentration did not (Table III).
The striking results obtained with thiourea and with thio- acetamide incorporated in water waxes (1.3 and 2.3 % decay, respectively, in treated fruit and 30.9 and 32.9% , respectively, in the checks, Table IV ) are believed due in part to the degree of penetration which this method o f waxing allows. Here the treated fruit is dried slowly by currents of warm air as it is con
veyed to the polishing brushes. B y the time it reaches the brushes, sufficient penetration has taken place so that removal of the excess thiourea or thioacetamide in polishing docs not impair control.
In the solvent-wax method the immiscibility o f thiourea solu
tion with the solvent requires that it be applied as a separate treatment. The fruit was dipped momentarily, drained, then rapidly brushed dry, and polished before exposure to the wax spray. Hence much of the tliiourea was removed before it could penetrate sufficiently and decay control was impaired (6.5%
decay in treated lots and 32.7% in checks, as is shown in Table II).
On the basis o f limited tests (two), if the treated fruit is air-dried before brushing and polishing, excellent control can be obtained with the solvent-wax method (1.3% decay in treated and 34.0%
in check, for a total o f 314 oranges). From the results obtained with these materials in conjunction with the water-wax and the solvent-wax processes, there is reason to believe that one or more of the compounds can be adapted for use with other fruit waxing and polishing procedures.
In order to remove all excess thiourea from the rind surface, in view of possible future tolerance limits, oranges were rinsed im
mediately after dipping in 5 % solution, then dried, polished, and waxed by the solvent-wax method. D ecay control was greatly decreased (Table V), and again the limiting factor appeared to be
insufficient time for penetration. There is evidence, however, that with longer immersion to permit adequate penetration, rinsing w'ould not materially lessen control.
Tests with thioacetamide, 2-aminothiazole, and quinosol were not so extensive as those with thiourea. In so far as the condi
tions of the tests with thioacetamide and with quinosol were similar to those with thiourea, the results were also similar.
The importance o f the relation between chemical structure and fungicidal activity warrants discussion. It was noted previously (8, 8) that thiourea, thioacetamide, and 2-aminothiazole are characterized by the presence of divalent sulfur and one or more amino groups in the molecule, and that in the case of thiourea and thioacetamide, at least, the presence of both is necessary for there is a constant oscillation between two possible structures, is uncertain. It is o f interest that the three most effective materials are highly water soluble, whereas many fungicidal compounds which are relatively insoluble in water have given little or no control of orange decays; in this connection Rivier’s observation (80) that thiourea tautomerizes to the thiol form only in water solution is noted. The fact that the sulfur in 2-aminothiazole is tied up in a rather stable ring structure m ay account in part for the mediocre control obtained with it.
It was noted that the very volatile thioacetic acid is highly toxic to all four fungi when in contact with them in vitro. When fruit was dipped in water solutions (1.25, 2.5, 5 % ) of thioacetic acid, however, no control was obtained. It m ay bo that the fungicidal activity of thiourea, for example, is due simply to the presence o f the sulfur but that the amino group is essential for
It was noted that the very volatile thioacetic acid is highly toxic to all four fungi when in contact with them in vitro. When fruit was dipped in water solutions (1.25, 2.5, 5 % ) of thioacetic acid, however, no control was obtained. It m ay bo that the fungicidal activity of thiourea, for example, is due simply to the presence o f the sulfur but that the amino group is essential for