Ethylene From Diseased Plants

C. E. Williamson, A. W. Dimock.

A number of symptoms of some fungus, bacterial, and virus diseases of plants are similar to those that develop on plants exposed to ethylene gas, the colorless, inflammable C₂H₄.

Those common symptoms include epinasty, or downward bending of leaves; yellowing; excessive overgrowths; retardation of growth; and premature dropping of leaves, flowers, and fruits. The development of such symptoms suggests the possibility that the diseased plant tissue may produce ethylene.

The role of ethylene gas in inducing various physiological responses in plants has been the subject of investigation for more than 50 years. The earliest recorded observation was made in Germany in 1864 on the toxic effects of illuminating gas on trees. The identification in 1901 of ethylene as the cause of the observed physiological effects of illuminating gas led to the accumulation of a large volume of information on the morphological, anatomical, and biochemical responses of plants to ethylene.

The citrus industry in California established the practice of degreening lemons with the products of incomplete combustion from kerosene stoves or the exhaust gases of internal combustion engines. In 1924 ethylene was identified as the constituent responsible for the loss of the green color.

Our present-day concept of fruit ripening, especially the sharp rise in respiratory rate with ripening termed the climacteric was developed in England from studies of apples in storage. The fundamental effect of ethylene is to initiate the climacteric rise in respiration. Emanations from ripening fruit will stimulate the onset of the climacteric in green fruit. The active emanation from ripening apples was identified by chemical analysis as ethylene in 1934. Subsequently, in all cases of physiologically active emanations investigated chemically, the active substance evolved has been shown to be ethylene. That is now so well established that the demonstration of a physiologically active emanation by any of the bioassay methods is a strong indication that ethylene is present in the gases evolved. In fact, F. E. Denny and Lawrence P. Miller, of the Boyce Thompson Institute, wrote in 1935: ". . . Any evidence that some other chemical is the principal factor must be accompanied by proof that ethylene was not present as an impurity in amounts sufficient to give a concentration of at least i part of ethylene in 20 million of air." For chemical identification, at least 25 to 100 parts per million by volume of ethylene is necessary. Where only small quantities of plant material are available or the amount of ethylene is extremely small, the chemical methods are useless. Bioassay methods thus become of paramount importance.

THE RESPONSE of various plant parts has been used to demonstrate the production of physiologically active emanations by plant tissues, especially by ripening fruit. Inhibition of sprouting of potato, inhibition of seed germination, and epinasty of sunflower, potato, and tomato leaves have all been used to detect ethylene. However, the triple response of etiolated pea seedlings, defined as a decrease in rate of growth in length and a swelling and horizontal placing of the region growing while exposed to ethylene, is the only bioassay method that readily yields quantitative results. The triple response has been demonstrated repeatedly to have distinctive qualitative and quantitative characteristics for different concentrations of gas. Etiolated Alaska pea seedlings are extremely sensitive and will respond to as little as 0.025 to 0.05 part ethylene per million of air. The triple response of etiolated pea seedlings has had widespread use since 1944.

Many species of fungi growing in culture on an artificial medium of one sort or another have been tested for the production of an active emanation. Rhizopus nigricans, baker's yeast, Diaporthe citri, Diplodia natalensis, Alternaria citri, Penicillium italicum, Sclerotinia sclerotiorum, Aspergillus niger, Oospora species, Alternaria species, Diplocarpon rosae, Cryptostictis caudata, Mycosphaerella ligulicola, Alternaria zinniae, and Histoplasma capsulatum (a pathogen affecting man) failed to give a positive response by bioassay methods. Penicillium digitatum, on the other hand, has been shown repeatedly to produce ethylene in abundance from a culture medium. Blastomyces dermatitidis and B. braziliensis, causal agents of blastomycosis, a lung disease of man, also produce ethylene from a synthetic medium.

The earliest report of an active emanation from healthy tissues is credited to a Captain Selfe, who observed that the gases from oranges packed in the hold of a ship were apt to cause premature ripening of bananas. His observations were verified in the laboratory by H. H. Cousins, who in 1910 reported: "It was shown, however, by direct trial that the emanations from oranges stored in a chamber were found to have the effect of bringing, about a premature ripening of bananas if these gases were passed through a chamber laden with this fruit."

Similar observations made by the United Fruit Company before 1917 showed that some bananas ripened more rapidly than the bulk of a shipment and this ripening tended to occur in pockets. The ripe pocket often acted as a starting point for the ripening of an entire bin of bananas. The ripe pocket was not caused by heat or carbon dioxide accumulation, although there seemed to be a direct correlation between respiration rate and accelerated ripening. A United Fruit Company chemist in 1923 reported the presence of an unknown gas in the respiration products of ripe bananas which caused accelerated ripening of green bananas.

In 1932 the volatile substances from ripe apples and pears were observed to inhibit the normal sprout development of potatoes. Healthy immature apple fruits were demonstrated in 1935 to produce ethylene in small amounts. When immature fruits were placed in closed containers, the accumulated volatile substances induced the climacteric, but similar fruits ventilated by a continuous stream of pure air did not ripen. Apparently a threshold value for the stimulating dose exists below which no effect is produced.

Denny and Miller reported a long list of plant tissues that produced an active emanation, as indicated by epinasty of potato leaves. The list included a wide variety of fruits and vegetables and the flowers, leaves, steins, seeds, and roots of other plants. Elmer Hansen, working with pears in storage in 1942, studied the relationship between ethylene production and respiration and observed that ethylene production was either greatly retarded or inhibited under anaerobic conditions, although little difference in the amount of carbon dioxide produced under aerobic and anaerobic conditions was obtained. Ethylene produced by Cornice pears increased steadily from 0.11 milliliter per kilogram per 24 hours at o C. to 0.19 at 10 , to 0.44 at 20 , then dropped to 0.33 at 30 , and ceased at 40 .

Investigators had observed as early as 1936 that the volatile combustible matter produced by orange fruits increased rapidly when the fruits became infected with Penicillium digitatum. Because the amount of combustible volatiles produced by P. digitatum growing on agar was very small, the rapid increase in amount of volatile combustible matter that occurs with the onset of fungal attack was assumed to be due to injury to the fruit and not to byproducts of the fungus. Similarly other workers found that when Golden Delicious apples in the post-climacteric state became infected with Penicillium and Botrytis, a rapid rise in the amount of volatile compounds occurred. No tests were made to determine the presence of ethylene in these cases.

The emanations of citrus fruits infected with Penicillium digitatum, Diaporthe citri, Alternaria citri, or Diplodia natalensis have been found to induce epinasty of the test plant leaves more quickly than the emanations of sound fruits. It was found also that the emanations of lemons infected with green mold, Penicillium digitatum, caused a rapid yellowing of green lemons and a shedding of the stem ends. The effects seemed to be due to ethylene. The emanations over a 24-hour period from a single lemon infected with P. digitatum contain approximately 0.064 milliliter of ethylene.

Recognition of the facts that diseased tissues produce considerably more ethylene than do healthy tissues and that a number of symptoms of common plant diseases are strikingly similar to the responses of healthy plants to exposure to ethylene gas led to the hypothesis that the symptoms of certain plant diseases may be caused by increased ethylene production. The rapid yellow coloration and early abscission of leaves infected by certain pathogens certainly suggest the action of a substance such as ethylene.

Two diseases characterized by such symptoms are the black spot of rose and the shot hole of cherry, caused by Diplocarpon rosae and Coccomyces hiemalis, respectively. An investigation was undertaken by C. E. Williamson to determine whether diseased tissues produced increased amounts of ethylene and, if so, to correlate the symptoms of the disease with the quantity of ethylene produced.

Different amounts of ethylene were indeed found to be produced by the different diseases, the amount apparently depending upon the pathogen involved. Large amounts of ethylene were produced by rose leaves infected with Diplocarpon rosae, cherry leaves infected with Coccomyces hiemalis, and chrysanthemum flowers infected with Mycosphaerella ligulicola. Somewhat less ethylene was produced by chrysanthemum flowers infected with Botrytis cinerea and rose leaves infected with Cryptosporella umbrina. A still smaller quantity, though significantly larger than that produced by healthy tissue, was produced by rose leaves infected with Sphaceloma rosarum and carnation foliage infected with Alternaria dianthi. In the other diseases studied, the infected tissue produced little more ethylene than did the comparable healthy tissue. Bean leaves infected with the halo blight bacterium, Pseudomonas phaseolicola, did not produce ethylene in a detectable quantity. In the black spot disease of rose ethylene production is at a maximum while the infected leaf is green, decreases as the leaf becomes yellow, and ceases when the leaf becomes brown. In those experiments, healthy leaves generally produced small quantities of ethylene, the amount depending to some extent upon the species of plant.

Plants infected with certain viruses were found by A. Frank Ross and Williamson to produce different amounts of ethylene. Large amounts of ethylene were produced by Physalis floridana plants infected with potato virus Y, provided the temperature of the greenhouse was such as to permit the development of local lesions and consequent necrosis or death of the tissues involved. Other viruses that produce local lesions on inoculated leaves were employed. They included tobacco mosaic virus (Marmor tabaci) on Nicotiana glutinosa, Datura stramonium, and Phaseolus vulgaris var. Scotia; alfalfa mosaic virus (Marmor medicaginis) on Phaseolus vulgaris var. Scotia; tobacco ring spot virus (Annulus tabaci) on Nicotiana tabacum var. Turkish; and potato virus X (Annulus dubius) on Gomphrena globosa and Nicotiana tabacum var. Turkish.