<* . ^° ^ V^ ^ ^ «£ v>>- 'C* A** *°V . * -?- p /% , > ^-_ ;0-A - W *2* v*V 0- ^ . * < $iv. -^ ^6* * ^ ^>v> ^ ,0 'V ^0* ' J> o° •-•- • ~0 * O „ ' .0 *v ^ .,-'• . ^ . : ^ & * 4+ ^°^ o V * ' v.* PHYTOPHTHORA DISEASE OF GINSENG A THESIS Presented to the Faculty of the Graduate School of Cornell University for the degree of DOCTOR OF PHILOSOPHY BY JOSEPH ROSENBAUM Reprint of Cornell University Agricultural Experiment Station Bulletin 363. October, 1915. PHYTOPHTHORA DISEASE OF GINSENG A THESIS Presented to the Faculty of the Graduate School of Cornell University for the degree of DOCTOR OF PHILOSOPHY BY JOSEPH ROSENBAUM Reprint of Cornell University Agricultural Experiment Station Bulletin 363. October, 1915. Tn exchaag* FEB 4- 1916 CONTENTS PAGE The host 65 The disease 65 Distribution 65 History and economic importance 66 Symptoms of the disease 66 Symptoms of other diseases which may be confused with Phytophthora. . 70 Alternaria blight 70 Sclerotinia white rot : 70 Sclerotinia black rot 71 Acrostalagmus wilt 71 Fusarium soft rot 71 Etiology 71 Early work on etiology 71 Pathogenicity 72 Identity of the organism 83 Examination of literature 83 Comparison of cultures 84 Macroscopic growth on various media 84 Kinds of spores produced on various media 85 Comparative morphology 87 Life history 89 Morphology of the fungus 94 Mycelium 94 Conidiophores 95 Conidia 95 Germination of conidia 97 Germination by germ tubes 97 Germination by swarm spores 98 Swarm spores 98 Sexual organs 99 Fertilization 100 Oospore 1 00 Oospore germination 100 Control 102 Spraying : 102 Removal of diseased parts 103 Deep planting 103 Rotation of crops 103 Sterilization of soil 104 Drainage 104 Bibliography 105 63 PHYTOPHTHORA DISEASE OF GINSENG Joseph Rosenbaum 1 THE HOST The American ginseng, Panax quinque 'folium L., is a member of the family Araliaceas. It was brought under cultivation about twenty years ago, but either the same or a closely related species has been cultivated in Korea for more than two centuries. According to Jartoux (1714) 2 , ginseng is a native of the North Tem- perate Zone. It .grows in rich, damp soils, such as prevail in hardwood forests. The Chinese ginseng is found principally between the 126th and 136th meridians, east longitude. The American species has about the same range of latitude, but extends farther south. The natural environment of the plant indicates three factors as favorable to its growth, namely, shade, good drainage, and an acid soil. The failure of the grower to take these factors into consideration when removing the ginseng plant from its natural habitat to his gardens has been primarily responsible for most of his losses. In 1S21 (U. S. Secretary of the Treasury, 1822) 352,992 pounds of ginseng were exported from this country and sold for $171,786, an average of nearly 49 cents a pound. In 19 13 (U. S. Department of Commerce, 19 1 4), the exports amounted to but 221,901 pounds. This was sold for $1,665,731, an average of about $7.50 a pound. This falling off in the number of pounds exported since the first shipments must be attributed in part to the diseases that attack this crop. It is becoming a common opinion among ginseng growers that the men who would grow ginseng successfully must first know all about the diseases to which the plant is subject. THE DISEASE DISTRIBUTION The disease known as mildew, Japanese mildew, or soft rot, attacks leaves, stems, and roots of the host. In this country it probably exists in every State in which ginseng is grown — Washington, Oregon, Nebraska, Kansas, Minnesota, Missouri, i\rkansas, Wisconsin, Michigan, Indiana, Ohio, New York, Pennsylvania, New Jersey, and Maryland. It is de- structive in Japan also, where it was first reported by Hanai (1900). It is known there as Koshi-ore, meaning bending at the loins. ' In making these investigations, the author had the cooperation of W. A. Orton, in charge of Cotton and Truck Disease Investigations, United States Bureau of Plant Industry. The author wishes to acknowledge also the many suggestions received during the progress of the work from Di. Donald Reddick and Professor H. H. Whetzel, of the Department of Plant Pathology, Cornell University. 2 Dates in parenthesis refer to bibliography, page 105. 65 66 Bulletin 363 HISTORY AND ECONOMIC IMPORTANCE Hori (1907), who was the first to study this disease rather carefully, states that it has long been known to Japanese ginseng growers. In the United States the disease was first discovered on ginseng in the State of Ohio by J. M. van Hook (1906), and it has since been observed more or less commonly in the ginseng-growing regions of the United States. Because of its general occurrence in ginseng gardens in many States, and the fact that it attacks all parts of the plant, both above and below the ground, it forms one of the most serious disease problems that confront the grower. PHOTOGRAPH BY WHETZEL FlG. 2. CHARACTERISTIC SYMPTOM OF GINSENG MILDEW One or more leaflets droop and hang limp and shriveled Hori (1907) reports a loss of $25,000, due to the disease, in one prov- ince of Japan in the spring of 1904. From observations by the writer in ginseng gardens for the past four summers, it is safe to say that in the eastern United States from twenty to thirty per cent of the plants are lost through the attacks of the disease before they reach the age of five or six years. SYMPTOMS OF THE DISEASE The tops of the plants are affected in a characteristic manner. Usually there is a drooping of a single one or all of the leaflets at the top of the Phytophthora Disease of Ginseng 67 petiole (Fig. 2). It happens in many cases that the disease attacks the main stem at the crown, or point where the leaf petioles are attached, and all the leaves droop and hang limp from the top of the stem (Fig. 3). Some other ginseng diseases exhibit similar symptoms, and microscopic Fig. 3. symptom of ginseng mildew in late stage The stage here shown is much later than that shown in figure 2. All the leaflets hang shriveled and dry examination and identification of the causal organism is often necessary in order to determine definitely what disease is present. The tissues at the point of infection are rapidly injured and lose their turgidity, and the leaflets hang limp from the petiole. 68 Bulletin 363 The leaf blades also show characteristic lesions or spots. The spots appear dark green and water-soaked, much like those of the Alternaria blight in its early stages. A week or two after the first appearance of the spot, the center becomes white, the margins remaining a dark water- soaked green. The spots vary in size from one centimeter in diameter to lesions involving the entire leaf. The demarcation between diseased and healthy tissue is not sharply defined. The spotting shows on both sides of the leaf, but in general the differently colored regions within the spot are better shown on the upper surface (Fig. 4). Fig. 4. lesions of ginseng mildew on leaves The water-soaked margin of the lesion, particularly in the leaflet at the right, should be noted In the early stages of the disease, especially on the. stalk or the petiole, the surface of the affected parts may show an almost indiscernible silvery white coating. During periods of sunshine the diseased tissues dry up quickly, leaving the dead and shriveled leaves at the top of the stem. Under such conditions the disease does not spread down the stem with great rapidity. If atmospheric conditions remain moist and cloudy, the entire stalk is gradually involved. On pressing a diseased stalk between thumb and forefinger it is found that, in contrast with the ordinarily firm stalk, the diseased one is hollow. The hollowing of the stem is preceded by a watery discoloration of the tissues. Phytophthora Disease of Ginseng 69 The roots may be attacked, showing a semi-soft rot. The lesion may- start at any point on the root and in a short time involve all the tissues. When such roots are allowed to remain in the soil for any considerable Fig. 5. SYMPTOM OF ginseng mildew on root The two roots have been cut longitudinally and show external and internal appearance. The root on the right was kept in a moist chamber for two days, and the external view shows well the growth of mycelium on the surface time, various organisms, such as Fusaria and bacteria, invade the diseased tissues, causing them to become soft. At this stage the disease is often accompanied by a disagreeable odor characteristic of vegetable decays. yo Bulletin 363 In some cases the disease starting in the root spreads up the stem. Roots cut longitudinally, showing the characteristic rotting, are illustrated in figure 5. When the disease originates in the root and does not extend rapidly into the stem, the leaves may take on various shades of yellow and red, resembling the colors that they naturally assume toward the close of the growing period. Such ' discoloration of the tops in the early part of the season, however, while indicating some pathological condition, is not to be associated necessarily with the Phytophthora rot. Any disturbance in the functions of the root may cause such discoloration, and this condition may occur in the case of other rots, as, for example, the Sclerotinia white rot (Sclerotinia libertiana Fckl.). SYMPTOMS OF OTHER DISEASES WHICH MAY BE CONFUSED WITH PHYTOPHTKORA The symptoms of a number of other ginseng diseases may be confused with those of the Phytophthora disease. Such are the Alternaria blight, the Sclerotinia white rot, the Sclerotinia black rot, the Acrostalagmus wilt, and an undescribed fusarial rot. ALTERNARIA BLIGHT Alternaria blight is a very widespread disease caused by a fungus of the genus Alternaria, designated by Whetzel as species panax, although a technical description has not been published. It attacks stems, leaves, and roots of the host. The first symptoms in the spring appear as dark brown cankers on the stem near the surface of the soil. The spots on the leaves are similar in size to those caused by Phytophthora, but may be distinguished from the spotting caused by the latter in that they exhibit a broad, rusty brown border. The Alternaria lesions may also appear on the top of the plant as the point where the leaflets are attached to the petiole or where the petioles arise at the top of the stem, as in plants affected with the Phytophthora disease. The lesions are, however, readily distinguished from those of Phytophthora in that they exhibit a velvety brown coating at the point of attack. The Alternaria disease also occurs on the roots in the form of a dry rot. The tissues of the root are shrunken, and are darker in color and firmer to the touch than roots affected by Phytophthora. SCLEROTINIA WHITE ROT Inoculation experiments have proved that the disease known as Sclerotinia white rot is caused by Sclerotinia libertiana Fckl. Affected plants wilt and sometimes fall over. This is due to a rotting of the stem at its base, which usually involves also the crown of the root. The roots Phytophthora Disease of Ginseng 71 become very soft and watery, but non-elastic. When placed in a moist chamber, an affected root invariably becomes covered with the white felt of the vegetative growth of the fungus. After a time numerous black sclerotia appear. sclerotinia black rot The cause of the disease known as Sclerotinia black rot is Sclerotinia panaris Rankin. 3 The roots only are affected. When the disease is in an advanced stage the entire root, as the name indicates, is coal black. In the earlier stages the rot is in all external symptoms similar to that of Phytophthora; any dissimilarity before the root has turned black can be detected only by the aid of a microscope. Sections through tissue affected with the black rot disease show an abundance of dark brown mycelium. ACROSTALAGMUS WILT The disease known as Acrostalagmus wilt is said by van Hook (1904) to be caused by a species of Acrostalagmus. The roots are the only parts attacked. The first external symptom is a wilting of the tops. The leaves finally become dry and papery to the touch. The limp appearance of the top at once suggests the possibility of a lack of moisture in the soil. Externally the roots show no lesions, but cross sections of affected roots exhibit to the naked eye a brown ring in the region of the sap tubes. FUSARIUM SOFT ROT The disease called Fusarium soft rot is caused by a species of Fusarium and is the most likely of these diseases to be confused with Phytophthora rot of roots. Roots attacked by the Fusarium are softer, however, and are always accompanied in the last stages by a strong odor. ETIOLOGY The cause of the Phytophthora disease of ginseng is a fungous parasite, Phytophthora cactorum (Cohn et Leb.) Schrot. The genus Phytophthora was founded by de Bary (1876) on the potato blight fungus, P. injestans. The genus as it now stands includes more than a dozen species. EARLY WORK ON ETIOLOGY The history of the study of the organism associated with the disease can be summed up as follows: Hanai (1900:28) and Hori (1907:153) demonstrated the constant association of the Phycomycete with the lesions of the ginseng leaves. Hori, in the article cited, makes the following statement: " Since this decaying process proceeds downward to the 3 Recent inoculations indicate that Sclerotinia panaris Rankin is probably identical with Sclerotinia smilarina Durand. 72 Bulletin ^63 roots, the entire plant begins to wilt and drops to the ground." No experimental evidence is furnished to show that the roots really rot, or that when this does occur it is due to the attacks of Phytophthora. Van Hook (1906) reports the constant presence of oospores of the same fungus in the stems of ginseng. Whetzel (19 10) appears to be the only one who has done any work on the pathogenicity of the fungus previous to the studies herein presented. In 1909 he made a series of inoculations, employing pure cultures of the Phytophthora isolated from ginseng, and reports that " in every case there was prompt infection, with the resulting lesions characteristic of the disease." Little stress is laid, however, on the Phytophthora causing injury to the roots. In a bulletin by Whetzel and the writer (19 12) the following statement is made: " Observations in the gardens show that the roots of plants, the tops of which are killed by the mildew, invariably rot unless promptly removed and dried." For the past four seasons the writer has been investigating the diseases of ginseng, studying for the most part the root rots. During the course of the work it has developed that a large proportion of the soft rot is due to Phytophthora. This bulletin presents a study of the life history and identity of Phytophthora as it exists on ginseng. PATHOGENICITY Several methods have been employed for isolating the fungus and growing it in pure culture. In 19 14 the organism was obtained from roots sent from a number of places — Ithaca, Scott, and Alden (New York), Kutztown (Pennsylvania), Mentor (Ohio), and Cassopolis (Michi- gan) . Isolation from diseased roots was successful only when made from roots on which the disease had just started. Later the lesion is invaded by other soil organisms to such an extent that isolation of Phytophthora is difficult or impossible. In most cases in which the fungus was isolated directly from the root, bits of tissue from the inside of the root at the junction of the healthy and the diseased areas were placed in tubes of oat agar. In some cases isolations were made directly from the root by placing the root in a moist chamber and carefully transferring to bean pod plugs the surface growth of mycelium which appeared in from two to three days. Pure cultures of the fungus are obtained most readily, not from the roots, but from the stems. The method of procedure is as follows: Diseased stems are immersed in a 1-1000 solution of mercuric chloride for from two to three minutes, the stems are split longitudinally, and tissue plantings are made on poured plates of oat or bean agar. In three or four days the fungus usually will have grown out from the bits of tissue, Phytophthora Disease of Ginseng 73 and subcultures can be made to test tubes of oat or potato agar. Good results may also be obtained, in case the disease is in its early stages, by washing the stems in mercuric chloride and placing them in a moist chamber. Conidia of the fungus usually appear within a period of from twenty-four to thirty-six hours. Transfers made from the surface growth usually give pure cultures. When the fungus is obtained in pure cultures it grows readily on a number of media. The writer has grown it on oat agar as made by Clinton, on Thaxter's hard potato agar, on corn meal agar, on sterilized ginseng stems, ,on a ginseng decoction, on bean pods, and on lima bean agar. 4 The growth on synthetic media has been very slight. After the ginseng fungus was isolated and grown in pure culture it was used for inoculating healthy plants. The experiments in inoculation of the tops have extended over a period of four years, but it was only in the spring of 19 13 that inoculation of the roots was attempted. Inoculation of the tops was made in the following ways: (1) The stems and leaves were sprayed with a water suspension of conidia and swarm spores, an atomizer being used for the spraying. The plants were covered with a bell jar for four or five days. Check plants were treated in a similar manner, with the exception that conidia were not added to the water. (2) By means of a platinum needle a bit of the fungus was removed from a pure culture and placed in the crotch of the plant. The inoculum was covered with moist cotton. For checks, the crotches of other plants were likewise covered with moist cotton. (3) By the use of a flamed scalpel a slight injury was made in the crotch of the plant and the inoculum was placed in the cut. Inoculation of the roots was made in the following ways: (1) The soil was removed from one side of the root. The root was slightly cut with a flamed scalpel and a bit of a pure culture of the fungus was placed in the cut. The checks were treated in a similar manner, but no fungus was placed in the cut. All the roots were then covered with soil. (2) Roots were placed in pots and were inoculated by watering with a solution containing conidia of the fungus. Previous to inoculation certain roots were punctured with a flamed needle. As checks, similarly treated roots were kept moist with water not containing the fungus. (3) Freshly dug roots were immersed in a solution of mercuric chloride for ten minutes, 4 The media used were composed as follows: Hard potato agar: 200 grams of potato, 20 grams of glucose, and 30 grams of agar, for every 1000 cubic centimeters of water. Oat agar was made according to the directions given by G. P. Clinton in the Report of the Connecticut Agricultural Experiment Station for 1909 and 1910, page 760. Corn meal agar was made according to the formula given by C. L. Shear and Anna K. Wood in Bulletin 252 of the United States Bureau of Plant Industry, page 15. Lima bean agar: 100 grams of ground lima beans and 15 grams oi agar for every 1000 cubic centi- meters of water. Bean pods: pods of ordinary string beans placed in test tubes with a small quantity of water and sterilized. 74 Bulletin 363 rinsed several times with sterile water, and placed in sterile test tubes. The inoculation was made in the test tube in much the same way as a subculture is made. The roots in the test tube were cut with a flamed scalpel. By means of a platinum needle bits of the pure culture were inserted into the cuts. The checks were likewise injured with the flamed scalpel, but no fungus was placed in the cuts. The test tubes were then placed in a moist chamber for from two to three days. Later this was found to be unnecessary. Results of inoculations on tops, on roots placed in the soil, and on roots placed in test tubes, are shown in figures 6, 7, and 8. It should Fig. 6. ginseng plants inoculated with a pure culture of phytophthora cactorum be stated here that when plants were injured, either on the tops or on the roots, a higher proportion of infection was obtained than when the tissues were not injured. As far as root rot in the garden is concerned, however, this makes very little difference, for one seldom finds a root in the soil without some injury due to rodents, insects, transplanting, or other causes. In all the inoculation work, in addition to the ginseng Phytophthora a culture marked Phytophthora cactorum was used. The latter was isolated by D. L. Peters, of Berlin, from Phyllocactus. This culture was found to be as pathogenic to ginseng as the Phytophthora isolated from ginseng. Phytophthora Disease of Ginseng 75 An attempt was made to determine whether the use of the different organs of the fungus for inoculating — mycelium, conidia, or oospores — made any difference in the ability to produce infection. These various organs were obtained from pure cultures of different ages and from different media. The mycelium was obtained from very young cultures on hard potato agar; the conidia were obtained from older cultures on the same medium, also from corn meal agar; the oospores, from old cultures on bean pods. No doubt mycelium was present in every case. No difference in ability to infect was found. The period of incuba- tion — ■ that is, from the time when the parasite is placed on the host to the time when the first visible FlG. 7. GINSENG ROOTS INOCULATED symptoms W i T h a pure culture of phytoph- appear — is thora cactorum r ,1 Growing roots two years old were slit with trom tnree a scalpel and inoculated with a pure culture , r- 1 of Phytophthora cactorum. The root on the tO nve QayS left was treated similarly but was not inccu- 1 1 lated. The inoculations were made on Oil rOOtS ana. August 4, 1913; the photograph was made from four to seven days later six days on tops. The length of time during which the fungus has been in artificial cultures does not seem to have any appreciable effect on its virulence. One culture isolated in the summer of 191 1 was employed, and another isolated in the spring of 19 13. They gave almost identical results in all the inoculation work. Wherever the rot was produced, it was always possible to make re-isolations by cutting out pieces of tissue at the boundary between the healthy and the diseased regions, and planting these on oat agar or bean pod Fig. 8. ginseng roots inocu- lated WITH A PURE CULTURE OF PHYTOPHTHORA CACTORUM Two-years-old roots of ginseng were disinfected, placed in sterile test tubes, and inoculated with a pure culture of Phytophthora cactorum. The root on the left was treated simi- larly but was not inoculated. The inoculations were made on May 15, 1013; the photogtaph was made ten days later 76 Bulletin 363 plugs. In a few instances the re-isolations were made by placing the rotted root in a moist chamber and then making plantings on oat agar from the mycelium appearing on the surface. Re-isolations from the tops were made by washing the stems for from two to three minutes in a 1-1000 solution of mercuric chloride, cutting them length- wise, and making plantings from the interior on oat agar. The fungus obtained from such re-isolations was identical with the original culture, as regards both morphology, behavior on culture media, and ability to produce the disease. Koch's rules of proof were carried out for the Phytophthora isolated from ginseng, and also for Phytophthora cactorum from Phyllocactus. In 1913 inoculations were made as shown in table 1. The percentage of infection obtained in all these inoculations leaves no doubt as to the conclusions to be reached. In 19 14 a greater number of inoculations were made and the results obtained were almost identical with those of 1913. TABLE 1. Inoculations Made in Various Ways with Phytophthora cactorum from Phyllocactus and from Ginseng. Summer of 1913 Date Source of organism used Condition of plants at time of inoculation Manner of inoculation Number of plants inocu- lated* Percent- age of in- fection* Number of plants used as checks f April 19 Phyllocactus and ginseng Four years old, begin- ning to push through soil Tops sprayed with sus- pension of spores 3 66.6 1 April 19 Phyllocactus and ginseng 1911 Same as above Plants not injured, inoculum placed in crotch 3 3 33-3 p -t 66.6G.J 1 April 19 Phyllocactus and ginseng 1911 Same as above Plants slightly injured, inoculum placed in crotch 3 3 1 00.0 P. 66. 6G. 1 April 19 Phyllocactus and ginseng 191 1 Same as above Soil removed, root injured, in< iculum placed in injury 4 100. April 19 Phyllocactus and ginseng 1911 Same as above Roots in test tubes 3 100. 2 *The number of plants inoculated and the percentage of infection are the same for Phyllocactus and for ginseng unless otherwise stated. t All checks remained healthy unless otherwise stated. t"P." indicates percentage for Phyllocactus; "G.," percentage for ginseng. Phytophthora Disease of Ginseng 77 Table i (continued) Date Source of organism used Condition of plants at time of inoculation Manner of inoculation r Number of plants inocu- lated* Percent- age of in- fection* Number of plants used as checks f April 19 Phyllocactus and ginseng 1911 Same as above Roots placed in pots and watered with a suspension of inoculum 3 April .19 Phyllocactus and ginseng 1911 Same as above Same as above, but roots pricked 3 3 66.6P.J 33 3G.J 1 April 20 Phyllocactus and ginseng 191 1 Same as above Roots in test tubes 3 100. 2 April 20 Phyllocactus Same as above Tops sprayed with a suspension of inoculum 3 33-3 1 April 20 Phyllocactus Same as above Plants injured, inoculum placed in crotch 3 100. 2 April 20 Phyllocactus Same as above Soil removed, root injured, inoculum placed in injury 3 100. 2 April 20 Phyllocactus Same as above Roots in test tubes 3 100. 1 April 20 Phyllocactus Saine as above Same as above, but roots pricked 3 2 May 16 Phyllocactus and ginseng 1911 Five years old, full- grown in garden Inoculum placed on leaves in drops of water 3 100. 1 May 16 § Phyllocactus Same as above Same as above 3 1 00.0 1 May 27 Phyllocactus and ginseng 1913 Same as above Same as above 3 3 1 00.0 P. 66 . 6G. 1 May 27 Phyllocactus and ginseng 1913 Same as above Tops sprayed with a suspension of inoculum 4 4 75. oP. 100.0G. 1 * The number of plants inoculated and the percentage o' infection are the same for Phyllocactus and for ginseng unless otherwise stated. t All checks remained healthy unless otherwise stated. t "P." indicates percentage for Phyllocactus; "G.," percentage for ginseng. § Inoculations were made on the same day as the preceding ones, but either in a different garden or in a different part of the same garden. Bulletin 363 Table i (continued) Date Source of organism used Condition of plants at time of inoculation Manner of inoculation Number of plants inocu- lated* Percent- age of in- fection * Number of plants used as checks f May 27 Phyllocactus and ginseng 1913 Same as above Plants slightly injured, inoculum placed in crotch 4 1 00.0 2 May 27 Phyllocactus and ginseng 1913 Same as above Soil removed, root injured and inoculated 4 100. 2 May 27 Phyllocactus and ginseng 1913 Same as above, but with roots removed Roots in test tubes 4 4 75-oP.t ioo.oG.f 2 May 27 Phyllocactus and ginseng 1913 Same as above Roots placed in pots and watered with a suspension of inoculum 3 1 May 27 Phyllocactus and ginseng 1913 Same as above, with roots pricked Same as above 3 1 June 13 Phyllocactus and ginseng 1911 Four years old, roots removed from garden Roots in test tubes 4 1 00.0 1 June 13 Phyllocactus and ginseng 1913 Same as above Same as above 4 100. 1 June 13 Phyllocactus Same as above Same as above 4 roo.o 1 June 13 Phyllocactus and ginseng 1911 Four years old, plants standing in garden Soil removed, root injured and inoculated 3 100. 1 June 13 Phyllocactus and ginseng Same as above Same as above 3 100. 1 June 13 Phyllocactus Same as above Same as above 3 100. 1 June 14 Phyllocactus and ginseng 1911 Same as above Plants slightly injured in crotch and inoculated 3 100. 1 *The number of plants inoculated and the percentage of infection are the same for Phyllocactus and for ginseng unless otherwise stated. t All checks remained healthy unless otherwise stated. % "P." indicates percentage for Phyllocactus; "G.," percentage for ginseng. Phytophthora Disease of Ginseng Table i (continued) 79 Date Source of organism used Condition of plants at time of inoculation Manner of inoculation Number of plants inocu- lated * Percent- age of in- fection* Number of plants used as checks f June 14 Phyllocactus and ginseng 1913 Same as above Same as above 3 100. 1 June 14 Phyllocactus Same as above Same as above 3 100.0 1 June 14 Phyllocactus and ginseng 1911 Same as above Soil removed, root uninjured, inoculum placed on surface 4 4 25.0P4 oG.J 1 June 14 Phyllocactus and ginseng 1913 Same as above Same as above 4 1 June 14 Phyllocactus Same as above vSame as above 4 1 July 1 iPhyllocactus and ginseng 1911 Five years old, plants standing in garden Tops sprayed with a suspension of inoculum 3 3 33-3P- 100.0G. 1 July I Phyllocactus and ginseng 1913 Same as above Same as above 3 66.6 1 July 1 Phyllocactus Same as above Same as above 3 66.6 1 July 1 Phyllocactus and ginseng 1911 Same as above Inoculum placed on uninjured leaves 3 100. 1 July 1 Phyllocactus and ginseng 1913 Same as above Same as above 3 100. 1 July 1 Phyllocactus Same as above Same as above 3 100. 1 July 1 Phyllocactus and ginseng 1911 Same as above Soil removed, root injured and inoculated 4 100. 1 July 1 Phyllocactus and ginseng 1913 Same as above Same as above 4 100.0 1 * The number of plants inoculated and the percentage of infection are the same for Phyllocactus and for ginseng unless otherwise stated. t All checks remained healthy unless otherwise stated. t "P." indicates percentage for Phyllocactus; "G.," percentage for ginseng. 8o Bulletin 363 Table i (continued) Date Source of organism used Condition of plants at time of inoculation Manner of inoculation Number of plants inocu- lated* Percent- age of in- fection * Number of plants used as checks f July 1 Phyllocactus Same as above Same as above 4 100. 1 July 1 Phyllocactus and ginseng 1911 Five years old, roots removed from garden Roots in test tubes 4 1 00.0 1 July 1 Phyllocactus and ginseng 1913 Same as above Same as above 4 100. 1 July 1 Phyllocactus Same as above Same as above 4 1 00 . 1 July 16 Phyllocactus and ginseng 1911 Three years old, plants standing in garden Plants slightly injured, inoculum placed in crotch 3 100. 1 July 16 Phyllocactus and ginseng 19 1 3 Same as above Same as above 3 100. 1 July 16 Phyllocactus and ginseng 1911 Same as above Roots not injured, inoculum placed on surface 3 1 July 16 Phyllocactus and ginseng 1913 Same as above Same as above 3 1 July 16 Phyllocactus Same as above Same as above 3 1 August 1 Phyllocactus and ginseng 1911 Four years old, plants standing in garden Soil removed, root injured and inoculated 4 4 75-oP.t 100. oG. % 1 August 1 Phyllocactus and ginseng 1913 Same as above Same as above 4 100. 1 August 1 Phyllocactus Same as above Same as above 4 100. 1 August 1 Phyllocactus and ginseng 1911 Four years old, roots removed Tom garden Roots disinfected and placed in test ;ubes 3 100. 1 * The number of plants inoculated and the percentage of infection are the same for Phyllocactus and for ginseng unless otherwise stated. t All checks remained healthy unless otherwise stated. X "P." indicates percentage for Phyllocactus; "G.," percentage for ginseng. Phytophthora Disease of Ginseng Table i (continued) Date Source of organism used Condition of plants at time of inoculation Manner of inoculation Number of plants inocu- lated* Percent- age of in- fection* Number of plants used as checks t August I Phyllocactus and ginseng 1913 Same as above Same as above 3 100. 1 August i Phyllocactus Same as above Same as above 3 100. 1 August i Phyllocactus and ginseng 191 1 Four years old, plants standing in garden Plants injured, inoculum placed in crotch 4 100. 1 August i Phyllocactus and ginseng 1913 Same as above Same as above 4 100. i August i Phyllocactus Same as above Same as above 4 100. 1 August 15 Phyllocactus and ginseng Same as above Plants not injured, inoculum placed in crotch 4 1 August 15 Phyllocactus Same as above Same as above 4 1 August 15 Phyllocactus and ginseng Three years old, plants standing in garden Tops sprayed with a suspension of inoculum 3 1 August 1 5 Phyllocactus Same as above Same as above 3 1 September 3 Phyllocactus and ginseng 1913 Same as above Roots not injured, inoculum placed on surface 4 1 September 3 Phyllocactus Same as above Same as above 4 1 September 3 Phyllocactus and ginseng 1913 Same as above Roots injured, inoculum placed on surface 3 1 00.0 1 September 3 Phyllocactus Same as above Same as above 3 100. 1 * The number of plants inoculated and the percentage of infection are the same for Phyllocactus and for ginseng unless otherwise stated. t All checks remained healthy unless otherwise stated. 82 Bulletin 363 Table i (concluded) Date Source of organism used Condition of plants at time of inoculation Manner of inoculation Number of plants inocu- lated* Percent- age of in- fection* Number of plants used as checks f September 3 Phyllocactus and ginseng 1913 Three years old, roots removed from garden Roots placed in pots and watered with a suspension of inoculum 3 1 September 3 Phyllocactus and ginseng 1913 Same as above Same as above, but roots pricked 3 1 September 3 Phyllocactus and ginseng 1913 Same as above Roots disinfected and placed in test tubes 4 4 50.0P.J ioo.oG.j 1 September 3 Phyllocactus Same as above Same as above 4 50.0 1 October 2 Phyllocactus and ginseng 1913 Four years old, roots removed from garden Same as above 3 100. 1 October 2 Phyllocactus Same as above Same as above 3 100. 1 October 2 Phyllocactus and ginseng 1913 Same as above Roots placed in pot, injured, inoculum placed on surface 3 100. 1 October 2 Phyllocactus and ginseng 1913 Same as above Same as above but not injured 3 100. 1 October 2 Phyllocactus and ginseng 1913 Same as above Roots placed in pot and watered with a suspension of inoculum 3 1 October 2 Phyllocactus and ginseng 1913 Same as above Same as above, but roots pricked 3 3 33 3 P- oG. 1 * The number of plants inoculated and the percentage of infection are the same for Phyllocactus and for ginseng unless otherwise stated. t All checks remained healthy unless otherwise stated. j "P." indicates percentage for Phyllocactus; "G.," percentage for ginseng. Phytophthora Disease of Ginseng 83 identity of the organism examination of literature A careful examination of the fungus from pure culture on various media shows that it resembles most closely Phytophthora cactorum (Cohn et Leb.) Schroeter (1889). Saccardo (1888) lists as synonyms of this species the following: Peronospora cactorum Cohn et Leb., P. Fagi Hartig, P. Semperuivi Schenk, Phytophthora omnivora de Bary. It may be well to briefly review here the history of the species. Lebert and Cohn (1870) observed a rot on Cereus giganteus and Melo- cactus nigrotomentosus . From their description of the symptoms it appears that the nature of the disease was a dissolution and separation of the individual cells, resulting in a more or less soft rot. An abundance of mycelium, conidia, and oospores was found. From the nature of the fungus Lebert and Cohn rightly determined that it was a Phycomycete, and described it as Peronospora cactorum. As far as they could deter- mine, haustoria were lacking. Hartig (1876:121) describes a fungus, Peronospora Fagi, as causing a disease of beech. A similar fungus was described the previous year by Schenk (1875), under the name Peronospora Sempervivi, found by him to be causing a disease of Sempervivum. De Bary (1881), in order to settle the identity of these forms — which he held to be identical — with little regard for priority or rules of nomenclature gave them collectively the name Phytophthora omnivora. Hartig (1882:42) wrote as follows in com- menting on this change: " I accept this new name, since it behooves me above everything else to desire a name for the parasite that shall bear no false significance. Since I have, from the earliest time, observed that the disease attacks maple, pine, larch, and fir, I believe omnivora is- better deserved than Fagi. In opposition to many systematists of later times who from their researches on priority have changed the customary name for another never accepted as valid by the science of the past one hundred years, I have been of the opinion that names existed, not for the authors, but for the scientific public." 5 De Bary was aware of the work of Cohn and Lebert when he wrote that there was not a doubt in his mind that the two forms were identical. He proposed the specific name omnivora as better expressing the nature of the organism. Schroeter (1889:236), recognizing the work of Cohn and Lebert, adopted the original specific name cactorum and simply classed the fungus in the more recently formed genus. The name as it stands, therefore, is Phytophthora cactorum (Cohn et Leb.) Schroeter. f Translation from the German. 8| Bulletin 363 Syn. Peronospora cactorum (Cohn et. Leb.). Bietr. biol. pflanz. 1 : 51-57. 1870. Peronospora Sempervivi Schenk. Bot. ztg. 33:690-693. 1875. Peronospora Fagi Hartig. Zeitsch. forst.- u. jagdw. 8:117-123. 1876. Phytophthora omnivora De Bary. Bot. ztg. 39:585-595, 601-609, 617-626. 1881. Phytophthora cactorum (Cohn et Leb.) Schroeter. Krpyt-fl. Schlesien 3:235- 236. 1889. The following is the original description as given by Lebert and Cohn (1870): " Mycelii tubi graciles nonnunquam torulosi ramosi, ramis angulo recto patentibus, haustoriis destituti. Stipites conidiophori tenues, in modum cincinni unilateraliter pauce-ramosi, sub apicibus ramorum coni- diferis non raro vesiculoso-inflati. Conidia in stipitibus pauca hyalina, ellipsoidea vel ovata, apice papilla prominente munita majuscula = 0,048 mm. (1/28-1/15 mm.). " Oogonia conglomerata membrana tenui marcescente munita, singula oosporam singulam exacte globosam episporio valido luteo-fusco pellucido laevi praeditam foventia, diametro = 0,024 mm. (1/40 mm.). " Habitat in meatibus intercellularibus parenchymatis variorum Cac- torum quorum morbum putredine quadam finitum efficit. Observ. hieme 1 868-1 869 in viridario excellentissimi ducis a Jacobi Vratislaviae. " Unfortunately it has thus far been impossible for the writer to examine the type material or to make isolations from the original host, and he is compelled to rely entirely on the above description and the descriptions of the later workers for the identity of this fungus. For comparison, a culture marked Phytophthora cactorum was obtained from the Bureau pour le Distribution de Cultures de Moisissures, of the International Association of Botanists in Amsterdam, with the information that it was isolated from Phyllocactus by D. L. Peters, of Berlin. COMPARISON OF CULTURES Since the Phytophthora of ginseng resembles the culture marked Phy- tophthora cactorum, a detailed comparison is here given. 6 MACROSCOPIC GROWTH ON VARIOUS MEDIA Phytophthora from ginseng and Phytophthora cactorum from Phyllo- cactus were grown on hard potato agar, oat agar, corn meal agar, bean pod plugs, and sterilized ginseng stems. Various synthetic media were also employed, but the growth on these was so small a^ to make them unsuitable for this work. On all the media no difference in the two forms was noticeable, either in rapidity of growth or in luxuriance of growth. Hard potato agar. — A white, fluffy, aerial growth was produced in five or six days on hard potato agar. The growth was profuse. 6 A comparison of the Phytophthora of ginseng was also made with nine other species of Phytophthora. The results of this study will be published in a subsequent paper. Phytophthora Disease of Ginseng §5 Oat agar. — The growth on oat agar was profuse and was beneath the surface as well as at the surface, making a slightly yellowish, mealy growth. Corn meal agar. — On corn meal agar there was a slight growth, mostly subsurface. Bean pod plugs. — The growth on bean pod plugs was aerial as well as embedded in the tissues of the pod. The growth was white, but was less fluffy than on hard potato agar. Ginseng stems. — On ginseng stems there was a very scanty surface growth. KINDS OF SPORES PRODUCED ON VARIOUS MEDIA The kinds of spores produced and the time of appearance of these are shown in table 2 : TABLE 2. Kinds of Spores Produced on Various Media At the end of two weeks Organism On potato On oat On corn meal On bean pods On ginseng stems Phytophthora from ginseng Numerous conidia Oogonia, oospores, conidia Few conidia Few oogonia Numerous conidia, few oogo- nia, few oospores P. cactorum from Phyllo- cactus Numerous conidia Oogonia, oospores, conidia Few conidia Few oogonia Numerous conidia, few oogonia, few oospores At the end of six weeks Organism On potato On oat On corn meal On bean pods On ginseng stems Phytophthora from ginseng P. cactorum from Phyllo- cactus Numerous conidia Numerous conidia Conidia, few oogo- nia, numer- ous oospores Numerous conidia, few oogonia, numerous oospores Few conidia, few oogonia, few oospores Few conidia, few oogo- nia, few oospores Numerous oospores and conidia Numerous oospores and conidia Numerous conidia, few oogonia, few oospores Numerous conidia, few oogonia, few oospores It was found in several series of the above that, while the time of ap- pearance of the different spore forms may vary for the different strains of Phytophthora, eventually the same spore forms appear on a given 86 Bulletin 363 medium. Oat agar and bean pods are especially favorable for the pro- duction of the sexual bodies. In the case of bean pods, the conidia appear mostly on the aerial growth, while the oospores are imbedded in the tissues of the pod. FlG. 9. MYCELIUM OF PHYTOPHTHORA, (a) FROM GINSENG, (b) FROM PHYLLOCACTUS The drawings were made from mounts of mycelium of the two oiganisms growing on oa.t agar, No con- stant difference can be noted Phytophthora Disease of Ginseng 87 COMPARATIVE MORPHOLOGY The morphological studies were made from cultures of the same age, grown on the same medium. No differences were noted in the mycelium of the two cultures (Fig. 9). In very young cultures there is a slight tendency to branch at right angles, but there is such great irregularity in the mycelium that such can hardly be taken to be a constant character. The conidia vary greatly in shape and size, but no difference between strains could be detected either when grown on the same or when grown on different media (Fig. 10). Over four hundred measurements were made for each form from cultures of different ages and grown on different Fig. 10. conidia of phytophthora, (a) from ginseng, (b) from phyllocactus The drawings were made from mounts of cultures of the two organisms growing on oat agar. Both drawings are made to the same scale media. The measurements most commonly obtained are the same for the Phytophthora from ginseng and for P. cactorum from Phyllocactus, 34-5 X 27M- Inoculations were made on freshly disinfected ginseng roots in test tubes and the two forms were re-isolated from the inoculated roots. Pedi- greed single-spore cultures were then made, and conidia and oospores were again measured. The same differences appeared as were shown previously. Different workers have given different measurements, though working presumably with the same species. This is brought out in table 3, in which the measurements given by individual workers for the same species are presented. Bulletin 363 TABLE 3. Showing Variation Given by Different Workers for Phytophthora cactorum Author Measurements of conidia ( micromillimeters) Diameter of oospores (micromillimeters) Cohn and Lebert Hartig Schenk De Bary Sehroeter Osterwalder Himmelbaur Zimmerman Hori / Bubak Van Hook Author 48 x 35-68 ^5-40 * 35-40 x 50-60-90 . . . 35-40 x 50-60 14.64-24.4 x 119.56 Not given 1 7-30 x 25-60 30-50 x 50-60 Abnormal 29 x 85 . 5 . 15-25 x 15-120 30-42 x 40-58 34-5 *27 20 (oogonium) 16-24! 24-30 . 24 (oogonium) 30-45 None found 26-28 Not given Not given * Measurements cf conidia given by Schenk are as follows: " Die kleinsten derselben sind 5, die grossten 3<> Theilstriche meines Zeiss'schen Mikrometers lang u'nd 4 bis 25 Theilstriche breit " f De Bary states that the oospores are in general from three-fourths to four-fifths the diameter of the oogonium. He gives the measurements of the latter as from 24 to 30 microns in diameter. The meas- urements given above for the diameter of the oospores were derived accordingly. It is thus shown that great variations may occur, apparently in the same species. De Bary, knowing of these variations as given by Lebert Fig. 11. the sexual process in phytophthora from ginseng Various stages are shown in the development of antheridium. oogonium, and oospores. Fertilization tubes are very evident in a number of cases Phytophthora Disease of Ginseng 89 and Cohn, by Hartig, and by Schenk, did not hesitate to place all forms in a single species. Likewise, Osterwalder (1906), notwithstanding the fact that his measurements do not agree with the others, does not con- sider this of sufficient importance to establish a new species. FlG. 12. THE SEXUAL PROCESS IN PHYTOPHTHORA FROM PHYLLOCACTUS More than four hundred measurements of oogonia were made for each form. Measurements of oospores taken from various media, and the cultures of different ages, show this spore form to be very constant. The measurements of the two forms are identical, being 27/i in diameter. The method of fertilization likewise does not differ in the two forms (Figs. 11 and 12). A summary of the above comparisons, taken together with the results of the inoculations as shown previously, proves that the Phytophthora isolated from ginseng is identical with the culture marked Phytophthora cactorum (Cohn et Leb.) Schroeter isolated from Phyllocactus. LIFE HISTORY Careful studies of the fungus have been made in order to determine its various relations to its host and the morphological characters of the parasite itself. These studies have been made with fresh material from the garden and with pure cultures on various media. Plants which early in the spring show characteristic drooping usually exhibit no external evidence of the fungus. They show a slight shrinkage and browning of the tissues of the stem, and often the shrunken tissue appears water- soaked. If the stems are placed in a moist chamber for a day, a silvery 4 o .' A ^ "" <* ,o o V °^ :-■ V it 9 . • . *^^ 4 o, ** ■ •<*> ' : - "^ '^cy £°* DOBBS BROS. LIBRARY BINDING 1APR 76^ ST. AUGUSTINE •^^32084 .o- 7 ^ '