JOURNAL OF AGRICDLTURAL RESEARCH

Voi,. VIII WASHINGTON, D. C, FEBRUARY 26, 1917 No. 9

A SQUASH DISEASE CAUSED BY CHOANEPHORA CUCURBITARUM1

By FREDERICK A. WOLF, Botanist and Plant Pathologist, North Carolina Agricultural Experiment Station

INTRODUCTION It was noted during the past season (1916) that summer squashes in the vicinity of West Raleigh, North Carolina, were attacked by a phy- comycetous fungus, Choanephora cucurbitarum (B. and Rav.) Thaxter. Attention was directed to this fungus primarily because of the luxuriance of its fructifications on affected parts of squashes {Cucúrbita spp.) and of the rapidity with which it destroyed such parts. A study was there- fore made of the development of the disease and of the morphology of the causal organism. Conidial, sporangial, chlamydosporic, and zygosporic stages have been developed, and there have been made certain observa- tions which are at variance with previously recorded facts relative to C. cucurbitarum and other species of the same genus. It is the present purpose, therefore, to record the results of this study as additional knowledge of this exceptionally interesting form and to indicate its importance as a parasite on the squash plant.

HISTORY AND DISTRIBUTION OF THE FUNGUS This fungus has long been known to occur within the United States, having first been described in 1875 by Berkeley2 as Rhopalomyces cucur- bitarum from decaying squashes collected in South Carolina by Ravenel. The organism has subsequently been found in other States, as noted by Thaxter3 in his account of the genus Choanephora. Dr. Thaxter himseíf collected the fungus on squashes at Waverley, Massachusetts, and on cultivated species of Hibiscus and a wild malvaceous plant at Eustis, Florida. He records the fact that the same plant is in the Curtis Her- barium at Harvard University under the name "Aspergillus cucur- biteus, Hillsborough, N. C, on squashes." He further examined speci- mens of the same fungus on squashes sent from New York by Prof. Peck and from Ohio by Prof. Morgan.

1 Published with the permission of the Director of the North Carolina Agricultural Experiment Station. 2 Berkeley, M. J. Notices of North American fungi. In Grevillea, v. 3, no. 27, p. 109. 1875. » Thaxter, Roland. A New England Choanephora. In Rhodora, v. 5, no. 52, p. 97-102, pi. 46. 1903.

Journal of Agricultural Research, Vol. VIII, No. 9 Washington, D. C Feb. 26,1917 kg . v Key No. N. C—3 (3I9) 320 Journal of Agricultural Research vol. vin, No. 9

Clinton,1 in 1903, reported the occurrence of the disease in Connecticut, and included photographic illustrations of the fungus on squash flowers. This species has also been noted in Brazil on the petals of a species of Hibiscus and on certain other plants, whence it was described by Möller 3 as Choanephora americana. Numerous collections have been made during the past summer in the vicinity of West Raleigh, N.C., and in two other localities within the State,—Winston-Salem and Walnut Cove. Since these points are rather widely separated, it is believed that Choanephora cucurbitarum is generally present throughout the State. From reports received, unaccompanied, however, by specimens, there seems to be no doubt that the disease was present during the past season in other Southern States. HOSTS OF THE FUNGUS During the past summer the disease was found on several varieties of Cucúrbita pepo, being most destructive on the "pattypan*' types of summer squashes, commonly known as cymlings. The fungus has also been found on the fading flowers of cucumber {Cucumis sativus) althea (Hibiscus syriacus), scarlet hibiscus (Hibiscus coccineus), okra (Hibiscus esculentus), and cotton (Gossypium herbaceum). It appears to be para- sitic, however, only upon the squash. Previous investigations, as has been indicated, reported the occurrence of C. cucurbitarum on squashes, Hibiscus spp., a wild mallow, and certain other plants. ECONOMIC IMPORTANCE The disease is probably not the cause of appreciable damage to squashes annually, but appears in epidemic form only under certain meteoro- logical conditions. Conditions of high humidity and excessive rainfall were generally prevalent in the Southern States during the past summer, and are believed to be correlated with epidemics of the squash disease. At no time during the four preceding summers has the disease been sufficiently destructive to attract the writer's attention. Since squashes are not a crop of any considerable financial importance to the market gardener in North Carolina, it is especially difficult to obtain figures on the loss due to this disease. The crop is very generally grown in home gardens, and even though a considerable proportion of the fruits decay, an ample crop for home use remains. All of the flowers have been observed to be blighted and all of the fruits destroyed on certain vines. Thaxter,3 in discussing the injury by C. cucurbitarum, says: "It is undoubtedly often responsible for the destruction of no inconsiderable percentage of the crop." Clinton4 states that "it no doubt often blasts the blossoms and may rot the fruit, also."

1 Clinton, G. P. Report of the botanist. Diseases of plants cultivated in Connecticut. In Conn. Agr. Exp. Sta. 27th Ann. Rept., [19021/3, p. 359, pi. 26. 1904. 2 Möller, Alfred. Phycomyceten und Ascomyceten. p. i8t pi. i( fig. 1-4. Jena, 1901. (Schimper, A. F. W. Botanische Mittheilungen aus den Tropen. Heft 9.) 8 Thaxter, Roland. Op. cit. * Clinton, G. P. Loc. cit. Feb. 26, i9î7 Squash Disease Caused by Choanephora cucurbitarum 321

APPEARANCE OF THE DISEASE The symptoms of the disease on flowers and fruits of squashes is quite characteristic. The fungus covers the affected parts with a luxuriant crop of conidiophores, with no evidence of any vegetative mycelium. The fructifications of two other fungi, Rhizopus nigricans Ehr. and Botrytis vulgaris Fr., may appear on the surface of decaying squash fruits, but neither of them possesses the metallic luster which charac- terizes the fructifying filaments of C cucurbitarum. The first evidence of attack on the flowers usually appears on the day following the opening of these flowers. Early in the morning of the second day, the furigus will have reached its most luxuriant development, especially immediately after a shower, and the fading flowers will be covered by a dense coating of immature, white conidial heads. Within a few hours these fructifications will have matured and will have changed in color from white to brown and at length to purplish black. When staminate flowers are attacked, the mycelium may pass downward into the flower pedicel, converting the tissues into a soft, translucent condi- tion. Abscission of the flowers generally occurs before the pedicels become invaded. The appearance of the disease on flowers and flower pedicels is shown in Plate 87, A. When pistillate flowers are attacked, the fungus passes from the fading flower into the young squash. Incipient decay from infection through the corolla is shown in Plate 871 Dy as is also complete decay involving the fruit in a soft, wet rot with a profuse envelopment of conidial branches. The decay of fruits progresses with remarkable rapidity, as is shown in Plate 87, B, C, The photographic exposure for figure B was made about 15 hours after the one for figure C, the squash, meanwhile, having been kept in a moist chamber. Occasional fruits were found in which the fungus appeared not to have gained entrance through the corolla.

ETIOLOGY AND DEVELOPMENT OF THE FUNGUS Choanephora cucurbitarum, the cause of this squash disease, is unique in that it is the only known American representative of the family Choanephoraceae. Two other species have been described from India, one C infundibulifera (Curr.) Cunn. on the flowers of Hibiscus rosa- sinensis and the other, C Simsonii Cunn. on Ipomoea rubro-coerulea and Zinnia elegans. The former of these was described by Currey * in 1873 as Cunninghamia, a new genus of mucedinous fungi, and was referred by Cunningham2 in 1879 to the Mucorineae.

1 Currey, Frederick, On a genus in the order of Mucedines. In Jour. Linn. Soc. [I^ondonl, Bot., v. 13, P. 333-334, pl. 7« î«73. 2 Cunningham, D. D. On the occurrence of conidial fructification in the Mucorini, illustrated by Choa- nephora. In Trans. I4nn. Soc. London, s. a, Bot., v. 1, p. 409-422, pl. 47. 1879. 322 Journal of Agricultural Research vol. vin, NO. 9

CONIDIAI, STAG3 Observations on the development of the conidial stage agree essen- tially with those of Thaxter.1 The erect conidiophores, whose com- parative size is shown in Plate 87, At are whitish at first, but at ma- turity change to a distinct metallic luster. They are broadest at the upper extremity, which becomes dilated into a capitate vesicle. From this capitulum, a few to a dozen or more ramuli arise, which in turn become vesicular (PL 85, A), The ramuli commonly remain simple, but occasionally branched ones are present. By a process of budding, an early stage of which is represented in Plate 85, B, the capitella become covered with a densely compacted layer of conidia (PL 85, D). The conidia vary in outline from oval to elliptical and are conspicuously marked by longitudinal striations (PL 85, C), some of which anastomose. They vary in size from 15 to 25 by 7.5 to 11 ¡x and in color from light brown to reddish brown. The base of each conidium is provided with a hyalin appendage, the adherent portion of the sterigma. When the ramuli are broken off, scars as shown in Plate 85, E> are evident on the primary vesicle. Variations from this normal course of development have been noted. Conidia may arise directly from the surface of the primary head, a condi- tion noted both by Thaxter 1 and myself and which is characteristic of the genus Rhopalomyces. Occasionally the conidiophore branches and gives rise to two primary heads on each of which a layer of conidia is formed. These are not necessarily depauperate forms, since they appear in cultures with normal well-developed conidial fructifications.

GERMINATION OF CONIDIA Germination readily follows when the conidia are sown either on nutri- ent agar, such as bean, potato, glycerin, or cellulose agar, or in drops of water. Cunningham 2 states that germination of conidia of C. infundí- bulifera does not occur in water. Germination of C. cucurbitarum does not proceed, however, with equal rapidity in water and in nutrient agar, as shown by Plate 85, G, H, the former of which represents growth on nutri- ent agar 3^ hours after the commencement of cultivation, and the latter growth in water, after an equal period. Conidia were obtained from the same source in both cases and were germinated under the same con- ditions of light and temperature. The first evidence of germination is the considerable increase in size which takes place before the emergence of the germ tube. Typically a single lateral germ tube is formed, occasionally two, which in some cases arise from the extremity. As a rule, the germ tubes will have emerged within two hours after sowing. The rate of subsequent development is indicated in the stages represented in Plate 85, /. Plate 85, /, a, shows

1 Thaxter, Roland. Op. cit. * Cunningham, D. D. Op. cat. Feb. 26,1917 Squash Disease Caused by Choanephora cticurbitarum 323 an early stage in the development of a hypha from each of two conidia which were kept under constant observation and outlined at 15-minute intervals, with the aid of a camera lucida. Each successive stage is thus 15 minutes older than the preceding one, making an interval of an hour between Plate 85, /, a, and Plate 85, /, e. Under favorable conditions of temperature (room temperature during July and August) the mycelium will have profusely covered a 10-cm. nutrient-agar plate within 24 hours and will have fruited luxuriantly. Thaxter1 found that this fungus fruits abundantly in a moist chamber when cultivated on squash tissues, but that fructifications very rarely appear on potato agar and similar nutrients in tubes. SPORANGIAI, STAGE. The sporangia of C. americana, which is regarded as synonymous with C. cucurbitarum, was described, as has been indicated, by Möller.2 Thax- ter states that he was never able to obtain sporangia of this organism, although he repeatedly cultivated it under unfavorable conditions of nutrition, which were held by Cunningham3 to favor their production in C. infundibulifera. Such unfavorable conditions are present when the artificial medium is exhausted by the vegetative growth of the fungus and when the mycelia developed from conidia produced in artificial culture. When grown under artificial conditions, C. infundibulifera appeared to lose its vigor progressively, since no reproductive structures developed in cultures the third generation removed from the natural host. Neither were sporangia formed on the host in the natural state, a condition simi- lar to that found by the writer with C. cucurbitarum. In fact, the conidial stage only has been found on squashes, the other stages having all appeared in culture. Sporangia generally appear with conidial fruits, but have been formed alone on one set of cellulose-, bean-, and glycerin-agar plates which were continuously kept in the dark. Other series of cultures on the same kind of media when grown in darkness developed a few of both types of asexual spores. A different strain of fungus was used in these series. Certain cultures on cellulose agar, when kept in the light, developed only sporangia. Evidently light alone is not the determining factor in the formation of conidia and sporangia in C. cucurbitarum. Cunningham3 never obtained sporangia apart from the conidial form. Mature sporangia are present as early as mature conidia. They usu- ally form most abundantly at the center of the colony on plate cultures. Sporangia are first evident as pendent, white, globular enlargements The sporangium becomes separated from the sporangiophore by a glob- ular columella, which can best be seen when mature sporangia are burst (PI. 85, L). At maturity the sporangia are black and vary in diameter from 35 to 160 /x. Diminutive sporangia have as few as two or three

thaxter, Roland. Op. dt. 3 Möller, Alfred. Loc. cit. » Cunningham, D. D. Op, cit. 324 Journal of Agricultural Research vol. vm, NO. 9 spores. The spores are ovoidal or elongated rarely inequilateral and of the same color as the conidia. They are somewhat larger than the conidia, being 18 »to 30 by 10 to 15 /x. They lack the striations which characterize the conidia, but are unique in that they are provided with two or three terminal or lateral hyalin appendages, from each of which a tuft of 12 to 20 hairlike processes extend (Pi. 85, N). These ciliate processes are from 1 to i^ times as long as the spores.

GERMINATION OF SPORANGIOSPORES

The germination of these spores is quite similar to that of conidia Within two or three hours after the spores are placed in nutrient media there is an increase in size of the spores, followed by the emergence of one or two lateral germ tubes (Pi. 85, /). Within a few hours a pro- fusely branched mycelium is formed, which in 24 to 48 hours has ma- tured a crop of sporangia and conidia.

CHLAMYDOSPORES

The mycelim in 4- to 5-day-old cultures on nutrient agar is commonly septate. Not infrequently well differentiated chlamydospores, as shown in Plate 85, 0, will have formed within this time. In cultures 2 weeks old chains of chlamydospores, as represented in Plate 85, M, are some- times developed. No chlamydospore formation occurred in the form which Thaxter1 had under observation. These bodies have never been observed to germinate. ZYGOSPORIC ■ STAGE Cunningham observed zygospore formation in the two Indian species of Choanephora, and his account of the development of C.infundibtdifera2 contains a detailed description of this process. Zygospores of C. cucur- bitarum appear not to have been observed prior to the present study. No noteworthy differences are presented by the American species when contrasted with the Indian forms. A club-shaped branch first arises from each of two adjacent mycelial filaments. These gradually enlarge and become densely filled with granular protoplasm. The contents tend to accumulate in the apical extremity, and a septum forms, separating the gamete from the suspensor. When two of these branches are sit- uated sufficiently close together, their bowed tips come in contact and the gametes become flattened along the opposed surfaces. The parti- tion is then absorbed and the two gametes fuse. Such a stage is repre- sented in Plate 86, B9 C, at which time the two saccular suspensors are quite clear and empty as contrasted with the immature zygospore, which is convex on the upper side and is filled with coarse granules and nu- merous yellowish oil globules. As the zygospore matures, this convexity

thaxter, Roland. Op. cit. 2 Cunningham, D. D. Op. cit. Feb. 26,1917 Squash Disease Caused by Choanephora cucurbitarum 325 is increased until the terminal walls of the suspensors tend to be in the same plane (PI. 86, £), and the suspensors appear to support it from one side. Meanwhile the exospore has thickened, become dark brown in color, and the oil globules have fused to make one large globule, which occupies one-third to one-fourth of the volume of the ripe zygospore. Zygospores vary in size from 50 to 90 /x in the longest diameter. . Mature zygospores were first observed in nutrient agar cultures 48 hours old which had been planted with conidia taken from squash flowers. They appeared, however, in only a small number of isolation cultures prepared in this way. They are formed embedded within the agar and on its surface and can be seen with the unaided eye as numer- ous black specks when the isolation plates are held against a white back- ground. Conidia and sporangia are always present in these plates. All attempts to germinate these zygospores have thus far met with failure. Furthermore, all attempts to determine the conditions necessary for zygospore formation have been futile. Curiously, no zygospores ap- peared in subcultures from cultures in which zygospores were present; nor have they ever been noted in culture except when the conidia for making these cultures were taken directly from the host plant. It was presumed that this might be due to a loss of vigor in artificial culture. It will be re- called that cultures of C. infundibulifera three generations removed from the natural host failed to form asexual spores. No such failure to form conidia and sporangia appears to occur in C. cucurbitarum, since it has been grown for 15 generations with no resultant decrease in the luxu- riance of its asexual fructifications. Numerous cultures have been obtained from single conidia and sporangiospores with the view of determining whether in this species zygospore formation was condi- tioned by heterothallicism. Thus far, however, no evidence of this separation of sexes has been found. When material for studying zygospore development was at hand, the press of other duties prevented the making of a detailed study of the process. Subsequently, it has been impossible to develop zygospores. However, several blocks of agar containing zygospores were fixed, sec- tioned, and stained. Unfortunately, only a few of the stages were ex- hibited in these sections. Plate 86, A, represents an early stage in which the gametes have been separated from the suspensors. Gametes, sus- pensors, and vegetative mycelium (Pi. 86, D) are found to be multinu- cleate. It is hoped subsequently to be able to furnish an account of the conditions necessary for zygospore formation and of the cytological phenomena accompanying this process. Since no complete account of all of the spore forms of this organism has ever been given, it seems desirable to present herewith a brief tech- nical description, together with its synonomy and the known list of hosts. 326 Journal of Agricultural Research vol. vm, NO. 9

Choanephora cucurbitarum (Berk, and Rav.) Thaxter Choanephora cucurbitarum (Berk, and Rav.) Thax., 1903, in Rhodora, v. s, p. 97-108, pi. 46. Rhopalomyces cucurbitarum Berk and, Rav., 1875, if» Grevillea, v. 3, no. 27, p. 109. Rhopalomyces elegans, var. Cucurbitarum Marchai, 1893, in Rev. Mycol., ann. 15, no. 57, p. xx. Aspergillus cucurbiteus Curtis, in Harvard Herbarium. Choanephora americana Möller, 1901, Phycom. u. Ascom., p. 18, pi. 1, fig. 1-4. Sporangia péndula, globosa, solitaria, polyspora, nigra, 35-160 p diam.; stipite nitido-violaceo; columella globosa; sporis ovoideis vel ellipsoideis, levibus, brunneo- rufis 18-30X10-15 M. utrimque interdum atque lateribus penicello ciliorum auctis; ciliis 25-45 M longis. Condidiophoris usque 6 mm. longis, nitido-violaceis; apice vescicula unica vel pluribus capitatis muriculatis terminatis; conidiis in vescicula capitatim msertis ovoideis longitrorsum striatis, brunneo-rufis, appendicula basilare hyalina, 15-25 X7.5-11 M. Chlamydosporae globosae vel oblongo-ellipsoideae. Rami zygosporae arcuati, zygosporis inaequilateralibus, único glóbulo olei genente, 50-90 n diam. Hab. in fructibusque floribus vivis Cucurbitae peponis ac in floribus languidis Hibisci esculenti, H.coccinei, H. syriaci, Gossypii herbacei, Cucumis sativii aliisque malvacearum, Americae Bor., atque in petalis adhuc stantibus Hibisci aliisque plantae partibus dejectis, Blumenau brasiliae.

PATHOLOGICAL ANATOMY Diseased tissues from squash fruits and flower pedicels were fixed, sec- tioned, and stained for this portion of the study. Mycelium was found to be present in all of the tissues that were involved in softrot or wetrot, with no evidences of disintegration in advance of the mycelium. It is both intercellular and intracellular, as shown by Plate 85, F, K, figure F representing the invaded tissue of the flower pedicel and K that of squash fruit. Parasitism appears to be of the necrotic type, with little or no transformation of the cell wall substance. Growth of the fungus on cel- lulose agar gave no evidence of the secretion of cellulase. Evidence of the presence of cellulase and cytase was further sought, with negative results in 14-day-old cultures of C. cucurbitarum on sterilized squash fruits. The fluid from these cultures was filtered through a Chamber- land filter and pieces of raw fruits were introduced under aseptic condi- tions into this sterile filtrate.

DISSEMINATION OF THE FUNGUS A microscopic examination of the inner surface of the corolla tube and of the filaments of the stamens revealed the fact that conidia of C. cucur- bitarum are commonly present in open squash flowers. Thirty of the 35 flowers which had opened on the morning of July 6, and which were examined in the afternoon of that day, contained conidia. On the after- noon of the 7th, conidia were present in all the flowers examined,—27. On the 8th, 7 flowers were picked before 7 a. m., and all had conidia within them. On the 10th, conidia were present in 25 flowers which had not yet closed. Since various species of bees so commonly visit squash flowers, à limited number were examined to determine whether they were instru- mental in distributing the conidia. The bees, when captured among the Feb. 26,1917 Squash Disease Caused by Choanephora cucurbitarum 327 squash plants, were placed in test tubes washed in a small quantity of water, and the wash water was examined by placing drops of this water on a microscopic slide. Conidia were quite commonly present in this water. The striped and spotted cucumber beetles (Diabrotica viitata and D. punctata, respectively) were also found to be carriers. The wind is also an agent in the dissemination of this fungus, as determined by a series of 13 agar plates which were exposed in a field of squashes on the morning of July n. On the following day C. cucurbitarium had developed on all of these plates. SUMMARY (1) Choanephora cucurbitarum is parasitic upon summer squashes, causing a blight of the flowers and a rot of the fruits. (2) The disease has been the cause of very considerable losses during the past summer. (3) Infection of fruits occurs, for the most part, by the passage of the fungus from the fading corolla to the young squash. (4) Various species of bees, striped and spotted cucumber beetles, and currents of air are agents of dissemination of the conidia. (5) The fungus has been found on languid flowers of cucumber, althea, okra, cotton, and other malvaceous plants. (6) C. cucurbitarum is the only known American species of this genus, (7) The conidial stage alone appears on affected parts of the several host plants. (8) Sporangial, chlamydosporic and zygosporic stages have been developed in artificial culture. None of these stages have previously been reported by investigators within the United States. (9) All of the reproductive stages have been noted in the same culture on nutrient agar. Conidia, sporangia, and zygospores may mature in cultures 24 to 48 hours old. No decrease in luxuriance of asexual fructi- fication has been found in culture on nutrient agar. PLATE 85 Choanephora cucurbitarum: A.—Immature conidiophore with ramuli developing on the primary vesicle. Xioo. B.—Budlike processes the beginning of conidial formation on the capitellum. Xioo. C.—Conidia. X250. D.—Mature capitulum covered with a layer of conidia. X125. E —^Primary vesicle from which ramuli have been detached. Xioo. F,—Mycelium in tissue of squash flower pedicel. X75. G.—Germination of conidia 1% hours old in nutrient agar. X250. H.—Germination of conidia 3^ hours old in water. X250. I.—a, Early stage in germination of conidia in hanging drop; b, growth of mycelium from same conidia as figure a drawn 15 minutes later; c> one-half hour later than figure a; ¿,15 minutes later than figure c; e, one hour later than figure a. X250. J.—Germination of sporangiospores. X250. K.—Mycelium of the fungus within the tissues of squash fruit. X250. L.—Variation in size of sporangia and columella. X70. M.—Chlamydospores borne in chains in old cultures. X250. N.—Sporangiospores with tufts of hairlike appendages. X2$6. O.—Chlamydospores formed singly in 4-day-old cultures. X250. (328) A Squash Disease Caused by Choanephora cucurbitaru

Journal of Agricultural Research Vol. VIII, No. 9 A Squash Disease Caused by Choanephora cucurbitarum PLATE 86

Journal of Agricultural Research Vol. VIII, No. 9 PLATE 86 Choanephora cucurbitarum; A.—Gametes prior to fusion. Gametes and suspensors are plurinucleate. X900. B.—Young zygospores whose exospore has not yet developed and whose contents are a dense mixture of granular protoplasm and small oil droplets. The origin from two mycelial branches is also shown. X320. C.—Lateral view of a young zygospore in the same stage of development as figure B. X320. D.—Ccenocytic mycelium. X900. E.—Mature zygospore with a well-developed exospore and a large oil globule. X 2 7 S • PLATE 87 Choanephora cucurbitarum: A.—Fructifications on squash corollas and flower pedicels. Natural infection. B.—Natural infection on squash fruit showing the rapidity of the decay when contrasted with figure C, which is reproduced from a photograph of the same fruit 15 hours earlier. C.—A diseased teratological squash. D.—Infection through the corolla in the fruit at the left and complete decay of the squash at the right. A Squash Disease Caused by Choanephora cucurbitarum PLATE 87

Journal of Agricultural Research Vol. VIII, No. 9