In?" ElQLOGICAL STUEMES OF DIAPQRTHE ‘J’EXANS {SACQ AND SY‘D.) GRATL 71143525 fer the aegis-er cf M. 5‘ MiCififififi STATE CQLLEE‘EE Edward Eugene Eufler 3948 THESIS This is to certify that the thesis entitled Biological Studies of Diaporthg Vexans (Sacc. and Syd.) Gratz. presented by Edward Eugene Butler has been accepted towards fulfillment of the requirements for M. S 0 __degree in_39_liafll__ Date _ “-795 (u... *-¢ BIOLOGICAL STUDIES OF ‘DIAPORTHE VEXANS (SACC. AND SYDJ GRATZ. by EDWARD EUGENE BUTLER A THESIS Submitted to the Graduate School of Michigan State College of.Agricu1ture and Applied Science in partial fulfillment of the requirements for the degree of MAS TER OF S CIENCE Department of Botany and Plant Pathology 19A8 THEmS II- III- IV. TABLE OF CON TENTS IntrOdUCtionOOOOo000.000.000.00no.0000.00.00 Rev1ew Of LiteratureOCOOOOO0.000.000.00.00000000000 A. HiStory and Nomenclature of the Fungus.......... B. Description of the Fungusxc................... 1- Vegetative Structures........................ 2. Reproductive Structures.......... Asema100000000000000 oooooooo semal... ..... 0.0.0.0000000000000 30 ’The organism in Pure CUltureoo...oo.oo.o 1+. Su8ceptible Hosts................. Experimental..... ....... .............. A. Materials and General Methods.......... ....... 1. Source of Isolates........................... 2. Single Spore Technique....................... 3. Infection ExperimentSOOOOO00000000000000.9000 B. Identification of the Organism............... C. Studies on Temperature and.Hydrogen—ion concentrationOOOOOOOO...0......00.0.0000....0... D. Effect of Light upon Growth and Pycnidial PrOductionCOOOOQQOOOQ0.....0.0.000.... E0 Carbon Utilizationooooo00000000000000.0000. Fo NitrOgen Utilization............................ Discuss1on...COO-OOOQQOOOOOOOOOIOOOQOOOOOOOOOOOOOOOC A. Experimental Methods.... ............ ............ B. Temperature and Hydrogen-ion Concentration...... C. Carbon Utilization................ 30753.1H Page 1 O\O\O\O\l\) 11 12 11+ 15 15 15 16 17 20 24 31 35 38 50 _‘-..-ux.-1- D. Nitrogen Utilization.................... ..... .... 58 0.000000000000000 63 E0 Light0000000000000000000000000 F. Practical ApplicationSOOOOCOOCO0.0.0.00.......O..66 V. Summary and Conclusions............................. 67 VI. Literature Cited.................. 7o Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table I. II. III. IV. V. VI. VII. VIII. IX- X. XI. XII. XIII. XIV- XV. LIST OF TABLES Summary of spore and pycnidial measurements of Diaporthe vexans recorded bv various WOI‘kGI‘B00.000000....00000000.00.0000.....000 Relative values of various media as given bv Palo (1936) for growth and pycnidial production of Qiaporthe vexans.............. Summary of spore measurements of the two strains of Diaporthe vexans used during the course of this study.................... Indicators used to adjust the hydrOgen-ion concentration of media...................... Growth of Qiapgrthe vexans after five days at various temperature and hydrozen—ion concentrations0......OOOOOOOOOOOOQOOOOOOOOO. The growth of Diaporthe vexans on various media in light and dark..................... Sources and quantities of carbohydrates employed in preparation of media............ Assimilation of galactose by Qiaporthe vexans during five days of growth at 26° 0.. Assimilation of lactose by his orthe vexans during five days of growth at 26° c......... Assimilation of sucrose by Dia 0 the vexans during five davs of growth at 265 C......... Assimilation of glucose by Diaporthe vexans 10 12 22 25 28 33 36 39 39 during five days of growth at 2 C......... 40 Assimilation of maltose by Dia orthe vexans during five days of growth at 56° C......... Growth of Qiaporthe vexans after five days at 26° C. using various sugars as a source or carbon.000000000000.00.00.0000.0.0.000... Sources and quantities of nitrogen employed in preparation of media..................... Growth of Qiaporthe vexans using various compounds as a source of nitrOgen........... 41 41 #5 46 LIST OF FIGURES Figure 1. Characteristic asexual spores and.hyphae of Qiaporthe vexans x1900. (A) Phoma type spores each with two guttulae (B) Stylo- spores and (C) hyphae from sterilized stems of SOlanum melongena........................ 21 Figure 2. Seedlings of Solanum melongena infected.with niaporthe vexans showing typiEal leaf spots 1012........................................ 23 Figure 3. Relation of hydrogen—ion concentration to the growth of Diapgrthe vexans at various temper- atures on a synthetic medium................ 29 Figure 4. Relation of temperature to the growth of niaporthe vexans on a synthetic medium at various hydfogen-ion concentrations......... 30 Figure 5. Effect of varying quantities of sugars upon growth of Diaporthe vexans.................. 42 Figure 6. Colony characteristics of Qiaporthe vexans on a synthetic medium containing various sugars in .12 mol. concentration............ 43 Figure 7- Effect of various sources of nitrogen on the growth of hiaporthe vexans.................. 47 Figure 8. Effect of light upon mycelial growth on potato dextrose agar, cornmeal agar, and a smthetic mediumOOOOOOOOOO0.0000.00.00.00... 48 Figure 9. Relation of light to the formation of pyc- nidia on cornmeal agar.................u... 49 \‘III a.“ ,- . gaudy shunts .AGKNOILEDGEMENTS I would.like to express my appreciation to all the members of the Department of Botany and.Plant PatholOgy whose cooperation made this work possible; especially Dr» C. J. Alexopoulos for his guidance, criticism, and advice given during the entire course of experimental work and.preparation of the manuscript. I would also like to thank Dr. Phares Decker, plant pathologist, University of Florida for supplying eggplant stems infected with the organism; and Ir. J. S. Tidd, Department of Research, Associated Seed Growers, Inc., for supplying eggplant seeds. E0 E0 B0 I. INTRODUCTION Present information reveals that Diaporthe vexans (Sacc. and.Syd.) Gratz is pathogenic only to the eggplant, Solanum melongena L. The organism is present wherever the eggplant is grown, sometimes causing infections that result in serious crap losses. All parts of the plant above ground -are susceptible to infection by Diaporthe vexans. The fruit is especially susceptible in the field, in transit, and in storage. The names most commonly used to designate the disease are damping—off, stem blight, leaf spot, and fruit rot, de- pending upon the plant part affected. Several articles, Harter (1914), Edgerton and.Mooreland (1921), Nolla (1929), and Palo (1936) have been written re- garding the symptoms and control of the disease and the taxonomy of the fungus. Existing literature evidences, how- ever a dearth of information relative to the physiology of Diaporthe vexans. ‘ This thesis will deal mainly with the "effector pH and temperature upon growth, and light upon the growth and spor- ulation of the organism. Studies have been included to as- certain the ability of the organism to assimilate various sugars and various forms of nitrogen. Little attention has been given to the symptoms, epiphytology, control and other aspects of the disease except where there is need in solving the problems which are purely mycological. II. REVIEW OF LITERATURE A. History and Nomenclature 2f the Fungus . Diaporthe vexans (Sacc. and Syd.) Grate has appeared in the literature since the late 19th century under the following names: 1891 Phone solani Hale. N. J. A r. 3&- Sta. 12th Ann. R.pt *lEEI, p.277. n_9_n. nud. acc.1895, 8111. Fuggorug, 11:#90. Not Cooke and Hark. 1884, in Grevillea, 13: 16. (fide Harter). 189ll§hLllosticta hortorum (not Speg.) . 1899 Phone vexans (Sacc. and Syd.) 8111. Fuggorum, 1#:889. 1905 Ascochyta hortorum (Speg.) C. 0. Smith. Delaware Agr- gggp. Sta. BuI. 70. 1914 PhomOpsis vexans (Sacc. and Syd.) Harter. Jour. Agr. 1333., 2:331-338. 1942 Dis-porthe vexans (Sacc. and Syd.) Grate. Phyt - Halsted (1891) reported a fungous disease arm melongena which caused a damping-off of eggplant seedlings in the hot-bed. He assigned M solani Hals. as the casual organism. He did not make a diagnosis but made reference to the fungus as folloaa: , ”The fungus that causes the death of the subject is so small that without the aid of a micro- scope nothing more is seen of it than a number of black specks embedded in the substance of the diseased portion." "The dark spots are spaces where the fungus threads have formed multitudes of spores inclosed in a dense mass of surrounding subs tance ." 1 Explanation given in text, p. 3, 5. Saccardo (1895). who evidently secured specimens from Halsted, gave the following brief morphological description of Hi___q__ma 591311; Hale: "... perithecia M, depressa. @- 19.1259.» 12.1.2 sperta; sporulae oblongae, stipitellatae.” Later Saccardo (1899) changed the epithet, 292..“ solani Hals., to £13959 vexans (Sacc. andSyd.) upon discovering that the name ngmg solani (Cooke and Hark.) had been previously employed for another organism. Related (1891), in addition to describing an organism causing damping-off of eggplant seedlings, also, attributed Phyllosticta hortorum Speg. as the causal agent of a fruit rot of eggplant. Referring to Phgllosticta hortorum he states, “The fungus which causes large brown and lifeless patches in the leaves is one that has been known for considerable time... ." Spegazzini (1881) described Phyllosticta hortorum as the fungus causing a leaf spot of Solemn nelongena in Italy. The pyc- nidia measured 80-90 microns in diameter and the conidia M x 2-2.5 microns . Soon after Spegazzini's diagnosis, Phyllosticta hortorum was assigned as the causal agent of a leaf disease of eggplant in America, with symptoms as indicated by Halsted. Smith (1904) described Ascochyta lympersici as causing a leaf spot of Solarium melongena. He stated that the fungus differed from flayllosticta hortorum in character of leaf spot, septation of spore, and size of spore . Smith noted that in material collected by Halsted the spores of Phyllosticta hortorum agreed in size with those given by Saccardo, i. e., #--6 x 2—2.5 microns, while those of the Ascochyta he found were 6-12 x 3.5—4 microns. The following year (1905), Smith described a leaf spot disease of eggplant assigning Ascochyta lycopersici Brun. as the causal organism. However, he noted changes in the char- acter of the spot, the number of pycnidia produced, and alter- ation of spore size from the previous year. The following statements made by Smith (1905) indicate that the fungus which he had under observation in 1903 and 1904 was possibly Phyllosticta hortorum.Speg. “A disease of eggplants has been described by Halsted of New Jersey which seems to be quite similar to this trouble. In fact, were it not for the fact that the fungus described in this paper has uniseptate spores I should regard them as identical. The septate spore char- acteristic does not always show plainly and.can be easily overlodked, and in fact, in some cases could not be found at all in.specimens that were examined." "The pycnidia containing the septate s pores are only found in old cultures and then not very abundant." "The writer is quite certain that the fungus described in America as Phyllosticta hortorum Speg. is the same as Ascochyta lchpersici Brun. He has never seen an authentically determined.specimen of Ascochyta lycopersici for comparison." Despite the apparently weak evidence, Smith preposed that the name be changed to Ascochyta hortorum (Speg.) Smith. However, since the new name was based upon somewhat uncertain taxonomic characters, pathologists in general did not accept the suggested change from Phyllosticta hortoru! Speg. to Ascochyta hortorum.(Speg.) C. 0. Smith. Harter (1914) believed that Phyllosticta hortorum, ghggg solani, and.Ascochyta hortorum were the same fungus. To ascertain the truth of his theory he conducted cross-inocula- tion experiments with.§hyllosticta hortorum and Phggg solani. His results showed that the two organisms were identical mor- phologically and that both organisms were capable of producing a fruit rot, stem blight, and.a rapid damping-off of eggplant seedlings. The symptoms produced on all parts of the eggplant by the two fungi were indistinguishable and.the incubation period was about the same. He also made thorough morpho- logical studies of fungi identified as Phylloaticta hortorum and ghgmg solani and in each case found the organisms to possess characteristics typical of the genus PhomOpsis. Harter, doubting somewhat, that Phyllosticta hortorum Speg. occurs in the United.States, sent typical specimens of the fungus to Spegazzini for examination. Spegazzini pointed out the main differences between his fungus and.the fungus forwarded bnyarter, stating that Harter's specimens were not Phyllosticta hortorum. Hence, Harter concluded that Phyllosticta hortorum had not been recorded in this country. Thus, the diseases of Solanum melongena attributed.to Phyllo- sticta hortorum, ghggg solani, and.Ascochyta hortorum from 1881 until approximately 1914 were in reality probably due to PhomoEsis vexans (Sacc. and Syd.) Harteru Harter believed that the results obtained by Smith (1904) (1905) were due to the presence of an Ascochyta and, at the same time, the so- called Phyllosticta hortorum. Edgerton and Mooreland (1921) found a species of piaporthe appearing frequently on agar cultures of Phgmopsis vexans, but they were unable to infect eggplant seedlings with the asco- spores. Grats (1942) found perithecia in six week old cultures of ghomOpsis vexans growing on 2% potato dextrose agar. ‘He was able to induce typical leaf and stem lesions by inoculating seedlings with suspensions prepared from crushed.perithecia, or with ascospore suspensions made from the exudate taken from the tip of the perithecial beaks. He did not, however, observe perithecia growing upon.the host. Based upon the results of these successful inoculations of eggplant seedlings with asco- spores, Gratz preposed the binomial Diaporthe vexans (Sacc. and Syd.) Gratz as the perfect stage of Phomoggis vexans (Sacc. and Syd.) Harter. B. Description 2; the Fungus. 1. Vegetative structures. Palo (1936) described the mycelium as consisting of "...fine hyaline, septate hyphae which ramify within the diseased.parts of the host tissues. The hyphae vary from 1.5 to 3.3 microns in diameter. In artificial culture some of the hyphal threads may grow as large as 4.2 microns in diameter." In general, hyphal meas- urements are not considered of taxonomic importance and are not given detailed consideration by other mycologists or plant pathologists. ' 2. Reproductive structures. Asexual:ASince the asexual reproductive structures are, perhaps, solely responsible for the prepagation of anporthe vexans they have been given much attention by those who have studied the organism. 7 The pycnidia were first described by Harter (1914) as ”irregularly shaped or flattened with a well developed beak." Edgerton and Mooreland (1921) state that the pycnidia are variable in shape depending upon the part of the plant upon which they grow and upon the amount of moisture present. They describe them, however, as flattened but sometimes more or less sub-globose, the top often prolonged into a beak. Palo (1936) describes the pycnidia as sub-globose but states that some may be lenticular-like or flask-shaped, while others are irregular and.beaked4 Nolla (1929) states that the pyc- nidia are typically beaked andwauotes the original description given by Harter. In his monograph of the genus Phomogsis, Diedicke (1911) states that the pycnidia of most species are flat when covered with the host tissue, becoming conical and beaked when exposed. They may, however, remain flat. Typically, pycnidia contain a single cavity, (Stevens, 1925), but Edgerton and.Moore1and (1921) have, upon occasion, found compound ones which often have more than one beak at the tap. They, also, found inclusions projecting into certain pycnidia from the base. In respect to the host tissue, the pycnidia may be innate or erumpent. Harter (1914) gave the following description: "0n the foliage and stems pycnidia loosely gregarious in more or less definite spots, on fruit compact, at first buried, later erumpent...." .According to Edgerton and Hooreland (1921) the pycnidia are generally embedded or partly so in the host tissue but if very moist conditions are present during develop- ment they may all be on the surface. Palo (1936) states that the pycnidia are first formed within the host tissues but as they grow in size they break through the epidermis. According to Nolla (1929) they are formed Just beneath the epidermis of the host, their beaks, when develOped, extending beyond the surface. Edgerton and Mooreland (1921) noted that beaks are usually better develOped on the pycnidia on old stems. They also give the pycnidial wall measurements as varying in thickness from 10 to 30 microns with the thicker portion of the wall at the base of the pycnidium. Harter (1914), on the other hand, fOund a ”thick, black wall at the tgp of the pycnidia becoming less noticeable at the base.“ The latter is given some confirmation by Diedicke (1911) who noted that a thickened sclerotia-like wall occurs typically at the apex of many species of Phomopsis. Considerable variation exists in the size of the pycnidia as reported by various workers. These differences are.re- corded in Table 1. Evidence points to the fact that a true ostiole does not exist in the pycnidia of Qiaporthe vexans. Harter (1914) makes no reference to a pycnidial Opening. Edgerton and Mooreland (1921) seem to consider the beak and pycnidial Opening as synonymous terms. They state "...the larger pycnidia may have more than one opening or beak at the tOp." According to Palo (1936) the pycnidia do bear ostioles; how- ever, he is not supported in this contention by other workers. In regard to the genus ghomOpsis, Diedicke (1911) states that various types of mouths are present among the species, and Clements and.Shear (1931) use "Pycnidia without ostiole" as a distinguishing feature. Also, Saccardo (1906) states that the pycnidia of the genus PhomOpsis are not regularly ostio- late. Two types of spores are formed within the pycnidia; short elliptical spores, Phoma or Phyllosticta type (so-called be- cause of the resemblance to spores of the genera Phyllosticta and.Phoma), and long narrow spores which often appear boom- erang-like (Fig. 1). The latter type are referred to by various authors as Septoria-type, Phlyctaena—type, and Stylo- spores. Harter (1914) described both types as follows: ‘ "...pycnospores subcylindrical, somewhat acute...continuous, hyaline, 2-guttulate, rarely 3...stylospores filiform, curved, rarely straight...." Edgerton and Mooreland (1921) refer to the elliptical spores as Phyllosticta type, describing them as small, elliptical in shape, single-celled, hyaline, and usually containing two prominent guttulae. Because of their apparent similarity to the genus Phlyctaena they applied this name to the filiform spores which they described as ”...hyaline, continuous and usually siCkle-shaped, though they may be nearly straight at times." The two spore types may be borne in separate pycnidia or tOgether in the same pycnidium Although various workers have reported variation in spore size (Table 1), they concur in regard to the morphology of the two types. Evidently Harter (1914) and Edgerton and Mooreland (1921) 10 :. a. \J n can as 1: I. \ u...\ no I O ...L I .1... \ t ‘7. “4 rl C‘\( I \3 rl‘ esseHSo seem +0f1rr-lp .\ \I.. \I‘ '“rPA(IHI'U QCKVQQIH n.m-e.fl A m1m c . t 1 :Hfioz 00 ca 0w 9: I msn+fiwu ma;H chacé on... CC... 1:31.11 .38 £135 ..\. , .Ji \ l . . _- ) .1» . 1 0 U V .\.' J1 +-\ n .I.‘ (( anlcmm (coined “1.2.1.... .1 mcpt A w 1m H V no o... x o e. s Q newline: OmHIULH ewrsu HAQHOHV occ c. means a mess H mu1ma its .on n.c-m s a- s1e1. . i cm s at as. 1 a .- 1 a. H m is see :-HpaeoH mmaoHaommem mmioi seaflom 1, -1 q mwrumkonrg : mDCHmMD wm ahum10 bum Hmfiz ..1i 411 1)). [knLvrrrer . r... omhpInSOMH k H H1H201rH (.1 Fresh .H mflfi E .9 11 did not experience difficulty in securing stylospores, but Nolla (1929) and Palo (1936) report that stylospores are not frequently found. There is general agreement, however, that stylospores are more abundant on old stems and.mummied fruit- Harter (1914) referred to the conidiOphores as "awl- shaped" and described them as simple, short, straight or slightly curved, hyaline and continuous. This stout, owl-shaped conidi- Ophore, according to Diedicke (1911), is characteristic of the genus PhomOpsis but, he states that only the Phomatype spores are borne upon these structures, the stylospores being borne upon shorter, conical shaped conidiOphores. Sexua1:-Gratz (1942), who prOposed the binomial Qiaporthe vexans for the perfect stage of PhomOpsis vexans (Sacc. and Syd.) Harter, described the perithecia, asci, and ascospores as follows: "...perithecia, occurring usually in clusters, were from 130 to 350 microns in diameter. The beak-like structures, ostioles, were carbonaceous, sinuate, irregular and from 80 to 500 microns long. The asci produced in these perithecia were 8-spore, clavate, sessile, 28-44 x 5-12 (av. 36 x 8.9) microns, hyaline, with thin walls, and apex slightly thickened and pierced by a narrow pore. The spores were biseriate, hyaline, narrowly ellipsoid to bluntly fusoid, quite uniform in size, 9-12 x 3.0-4.4 (av. 10.8 x 3.7) microns, bicellular, constricted at the septum with each cell usually containing two guttulae. In all respects, this fungus, isolated on two occasions from eggplant stems exhibiting characteristic symptoms of ”tip over," from Marion County, Florida, is, in appearance, a typical Diaporthe.“ 12 3. The Organism $212253 Culture. 'Qiaporthe vexans is not nutritionally fastidious, growing well on most ordinary culture media. Palo (1936) grew the organism upon a number of culture media. These are listed in Table II with the relative ability of each to produce pycnidia and to support mycelial growth. His investigation also showed that growth on all agar media was white, distinctly lobed at edges of colonies, and in some cases distinctly zonate. He also re- ports that growth was flat in general, but aerial tufts of hyphae were usually produced on papaya and potato dextrose agar. TABLE II. RELATIVE VALUES OF VARIOUS MEDIA (Palo 1935) FOR PRODUCTION OF PYCNIDIA AND MYCELIUM. Medium Mycelium Pycnidia Steamed corn meal a Steamed rice Steamed eggplant stems Steamed string beans Papaya agar Potato dextrose agar Oatmeal agar Prune agar Eggplant agar String bean agar Corn meal agar Cucumber seed agar Leonian's m. extract agarb UU UH-‘l-‘NUUUkk UHFHUUU‘NNk-‘Pkk a (1) Poor; (2) Fair; (3) Good; (4) Excellent. b KHQPO -1.2 gr., M 04-.6 gr., Peptone-.6 gr., Maltose-6.0 gr., aét extract- .0 gr., Distilled Ego—1000 cc, Agar 1.5-2.0 . 13 Howard and Desrosiers (1941) inoculated sterile vegetative structures of a large number of field and garden crOps and found.that the fungus would grow on all substrata tried. They also found that pycnidial production was greater on cauliflower petioles, carrot roots, and beet roots than on eggplant tissues. Nolla (1929) grew the organism upon oatmeal agar, corn flour agar and 1% dextrose nutrient agar. He reported that luxuriant growth was obtained with the latter medium, filling a dish, 90 mm. in seven days; and that he obtained poor growth upon oatmeal agar and corn flour agar. Nolla states that “Stromata began to develOp in these media on the fifth day and large numbers had appeared on the twentieth day. The stromata are black. One to several pycnidia arise in each stroma in these media. The pycnidia are typically beaked." Harter (1914) agrees that the fungus forms a stroma in culture and produces beaks 1 mm. or more in length. Edgerton and Mooreland (1921) found that the fungus pro- duces both kinds of spores in culture and Harter reported stylospore formation on sterile corn meal. The investigations of Nolla and Palo (1936), however, showed that stylospores were almost lacking on agar cultures although the Phoma-type were produced abundantly on many media. There is no record of stylospore germination. Edgerton and Mooreland give the optimum temperature for growth as ”around 29° C." but state that good growth occurs be- tween 21 and 32° C. Chupp (1925) gives "approximately 859 F." (29.4° C.) as the Optimum temperature, remarking that it will 14 grow at slightly higher and considerably lower temperatures. Palo (1936), who did his experimental work at Manila, Philip- pine Islands, incubated his cultures at room temperatures. The average temperature at Manila for the months of January, April, July, and October over a period of 47 years was recorded as 26.80 C. (Yearbook of Agr. U.S.D.A., 1941). Edgerton and Mooreland (1921) found that the fungus grows well on slightly acid or slightly alkaline media but not on strongly acid media. Nolla (1929) stated, ”We have not found much variation in our fungus." He discovered two kinds of spots on the host; from each type of lesion not less than fifteen isolations were made and compared on three media. Nolla did not observe dif- ferences that would indicate the presence of two strains. Edgerton and.Moore1and, however, after studying a hundred or more different cultures, noted variation in manner of infection, rate and.manner of growth and in ratio of the two kinds of spores. 4. Susceptible figgtg. As pointed out earlier, the egg- plant (Solanum.melongena L.) appears to be the only known host of Qiaporthe vexans. In cross inoculation experiments the following plants were inoculated with spore suspensions of the organism giving negative results: ‘LXQOpersicon esculentum, Capsicumannuum, Datura tatula, Ipomoea batatas ' (Harter, 1914), Solanum indicum, Solanum pyroconthum, Solanum mammosum (Howard.and.Desrosiers, 1941), and Solanum tuberosum 15 (Edgerton and Mooreland, 1921). The latter authors inoculated unknown species of Solanum.but they were unable to induce infection. III. EXPERIMENTAL A. Materials Egg General Methods. 1. Source 2; Isolates. Cultures of Qiaporthe vexans used for experimental work were isolated from fruit and stems of diseased.specimens of Solanum melongena. » Stems of Solanum melongena bearing many pycnidia of Qiaporthe vexans were obtained from fields near Gainesville, Florida. The fungus was isolated from these stems and grown in pure culture. From this culture a single spore was iso- lated which produced the strain used throughout the course of this work. A second strain similarly isolated from fruit purchased on the local market was carried 12.21122 but not used in each experiment. The strain isolated from stems will be referred to in this paper as A and that from the fruit as B- A section of stem, 50 mm. in length, heavily infested with pycnidia, was immersed in a l-lOOO solution of mercuric chloride. At the end of 30 seconds the section was removed and rinsed twice with sterile distilled water. Individual pycnidia were removed with a dissecting needle under low magnification of a binocular dissecting microscOpe. Five pycnidia were placed in a 4 c.c. sterile water blank and crushed with a sterile glass rod. A 4 mm. wire loop of the 16 resulting Spore suspension was added to each of 15 tubes con- taining 10 c.c. of potato dextrose agar1 which had been cooled in a water bath to approximately 42° C. The tubes were ro- tated rapidly to disperse the spores and the agar snapension was poured into Petri dishes, and incubated at 26° C. Eggplant fruits showing typical rotting but not fruiting bodies of the organism were also used for isolation purposes. An attempt was made to free the surface of the fruit from contaminating organisms by the following method: the fruit was washed thoroughly with a solution of sodium lauryl sul- fonate and.rinsed with sterile distilled water. The surface was then flooded with a 1-50 solution of 10% Roccal (Alkyl- dimethyl-benzyl-ammonium chlorides) by using saturated cheese cloth. The disinfectant was removed after three minutes with sterile distilled water. The fruit was then cut into two parts with a sterile scalpel and small pieces of diseased tissue removed and placed directly on potato dextrose agar in Petri dishes. Ten plates were prepared in this manner, two tissue sections per plate, and incubated at 26° C- Pure cultures of the organism were obtained by the two methods just described. 2. Single S2233 Technique. A method similar to that outlined by Thom and Raper (1945) was used to isolate single spores for preparation of stock cultures. A single pycnidium, from a culture on potato dextrose 1 120 gr. potatoes, 20 gr. dextrose, 18 gr. agar, 1000 c.c. H20- 17 agar, was crushed aseptically with a glass rod in a test tube containing 2 c.c. of sterile distilled water. Subsequent dilu— tions were made in 4 c.c. sterile water blanks until a 2 mm. transfer 100p contained an average of one to three Phomatype spores. The Van Tieghem cell provided an excellent aid for observing each loop of suspension under high-power of the microscOpe. The drOp of spore suspension was placed near the center of a clean cover glass and inverted over the glass ring. Addition of petroleum Jelly to the upper and lower edges of the ring served to prevent evaporation of the liquid. A 2% agar solution was cleared by the method given by Riker and Riker (1936) and a plate was poured containing 10 c.c. of agar. 0n the bottom of the Petri dish several circles (approx.‘7 mm. in diameter) were drawn with a wax pencil. A drOp of spore suspension was then placed on the agar above each circle and the plate incubated at 26° C. (At four hour intervals the area within each circle was searched under low- power of a microscOpe for germinating spores. When a circle was found containing only one germinating spore, the location was marked with ink on the surface of the plate. The section of agar above the ink was removed with a small razor-blade-type scalpel and transferred to a freshly poured plate of cornmeal agar. The presence of a single germinating spore was confirmed under low-power of a microscope. 3. Infection Experiments. Seedlings of Solanum melongena used for inoculation purposes were grown in a sandy soil in four-inch clay pots. The pots and soil were sterilized in an 18 autoclave for two hours at 15 pounds pressure. The seed, before planting, was treated with tetramethyl thiuram disul- phide (arasan) as a protectant against seed-borne infection. Three separate inoculation experiments were conducted. In the first, a five millimeter cube of agar from a rapidly growing culture of the organism on potato dextrose agar was placed near the middle of each seedling leaf. Each leaf was previously punctured with a sterile dissecting needle at the point where the agar was to be placed. The control plants were treated in the same manner, a five millimeter cube of sterile potato dextrose agar being placed over the puncture. Eighteen 24-day-old.seedlings were used; nine were inoculated, and nine were used as control plants. The pots bearing seed- lings (three per pot) were placed in a 12-inch clay dish con- taining one-half inch of water. The pots were covered with a bell Jar and incubated in the greenhouse. The bell Jar was removed after 48 hours. Temperature was recorded with a Taylor thermograph. Thirty 21-day-old seedlings, grown under the same con- ditions as those for the first experiments, were used in the, second tests. Each pot contained three seedlings; 21 were inoculated and nine were used for control plants. A spore suspension in sterile distilled water was prepared by crushing pycnidia grown on sterilized carrot roots. Five c.c. of the supernatant fluid containing the spores was aseptically pi- petted into a sterile atomizer Jar. After establishing the fact that spores were being released from the atomizer, the 19 . seedling stems and leaf surfaces were sprayed with the sus- pension. The control plants were Sprayed with sterile dis- tilled water. The pots were then placed in 12-inch clay dishes, one per dish, containing one-half inch of tap water and each covered with a large lantern-chimney type glass, moist chamber. The sides of the chambers were lined with water-saturated paper towels; petroleum Jelly was applied to the upper edges and the tsp covered with a glass plate. The seedlings were incubated in the greenhouse without artificial light at 21° C. .At the end of 48 hours the glass plates were removed. I In the third experiment 15 pots were used, each contain- ing three 30-day-old seedlings. Thirty seedlings were inocu- lated and 15 were used as control plants. Essentially the method was that employed in the second experiment with the following modifications. The spore snapension was applied to both upper and lower leaf surfaces (not injected) by using a hypodermic needle and syringe. The stems, however, were in- Jected about one inch from the soil line with a small quantity of the suspension. The glass plates covering the chambers were removed after 48 hours but replaced 24 hours later so that there was a one-inch Opening at the t0p of the chamber. At 12 hour intervals the seedlings were Sprayed with sterile distilled.water to increase the humidity. The saturated paper toweling was removed at the end.of 96 hours. The pots were incubated at an average temperature of 27° C. and sub- Jected to continuous artificial light from two 500 watt bulbs 2O placed three feet above the pots. The pots were arranged in three rows of five pots each. B. Identification 92 the Organism. Following are the characteristics of the organism ob- served in pure culture and on the host which were used to identify the fungus as Diaporthe vexans (Sacc. and Syd.) Gratz. When grown in the dark on potato dextrose agar, cornmeal agar, prune agar, and rice agar the organism pro- duces a white, somewhat lobed, zonate colony. On most natural media the mycelium is flat; however, numerous aerial tufts occur on potato dextrose agar. This compares favorably with the description and plates given by Palo (1936). Stro- mata are produced in culture, each tearing from one to several pycnidia (Nolla, 1929). The pycnidia are subglobose to beaked (Harter, 1914) with the latter type appearing more frequently in older cultures. The morphology of pycnidia occurring on the host is similar to that observed in pure culture. Phoma-type Spores (Fig. l,A) and conidiOphores similar to those described by Harter (1914) were found in all material examined on the host and in culture. However, stylospores (Fig. 1,B) which are said to be characteristic of the genus gnomOpsis (Harter, 1914), were found only in cultures of the organism growing on sterilized, four-months old eggplant stems. The spore measurements compared favorably with those given by Harter (1914) and others (Table I). These measure- ments are given in Table III and are based upon 50 Phoma- type spores and 25 stylospores. All measurements were made 21 nouns u. - (A) PYCNOSPORES (e) STYLOSPORES (C)HYPHAE I 22 at x960 using Spores from pycnidia formed on sterilized egg— plant stems. Several hundred spores from several pycnidia were observed in order to establish the range of size. Stylo- spores were not observed in strain 8- TABLE III. SUMMARY OF SPORE MEASUREMENTS (IN MICRONS) OF THE STRAINS USED DURING THE COURSE OF THIS STUDY. Phoma-type‘Spores Strain Stylospores .Average Range A 1-108 X 16-25 204 X 508 408'6 09 X 109-302 B Not observed 2.2 x 6.1 4.8-7.8 x 1.8-3.0 In order to establish definitely that the organism in question was Qiaporthe vexans, a series of three inoculation experiments were conducted with three-week-old seedlings of Solanum melongena. The first two experiments conducted at an average temperature of 21° C. were unsuccessful, the typi- cal leaf spot or stem blight disease failing to develop at the end of 30 days. In the third experiment, conducted at an average temperature of 27° C. disease symptoms appeared at the end of five days and all inoculated seedlings showed visible signs of infection at the end of 10 days. Strain A and strain B were used, both producing typical symptoms. 0n the leaves the disease first appeared at the end of five days as small circular, grayish-brown spots (Fig. 2), in many cases surrounded by a circular water-soaked area. These spots enlarged rapidly causing necrotic areas, curling, yel- lowing, and drOpping of leaves. Necrotic areas appeared on 23 Figure 2.-Seedlings of Solanum melongena seven days after inoculation with a spore suspension of Diaporthe vexans. (A) and (T) show yellowing of leaves and typical grayish-brown spots. Lesions present on (S) were not revealed in photographing. Plant (G) was not inoculated. 24 the stems at points where Spores were injected. These lesions were brown and elongate. (At the end of seven days the stems were not girdled. The organism was isolated from the typical lesions and grown in pure culture. Under the conditions of the experiment, pycnidia were not observed on the hOSt. However, when surface- sterilized, diseased leaves were placed on potato dextrose agar and incubated at 26° C., numerous characteristic fruiting bodies developed after 48 hours on the leaves. C. Studies 2g Temperature and.§ydrog§n-ion Concentration. The effect of temperature and hydrOgen-ion concentration upon growth was determined by using as a criterion the mean diameter of colonies on a synthetic medium at varying pH and temperature levels. A modification of Coon's (1916) synthetic solution was used, with 15 grams of agar added per 1000 grams of water. The formula is as follows:1 SUCI‘OSe.................................. 7020 are Dextrose...00000.0.OOOQOOOQOOOQOOOOOOoogo 5060 gr. ”@04000000000oeeeeeoeeooeeeoeeewe...coco 1023 are KH2POl+ooeeewe...00000000000000.0000000000 2072 61'. ENG o.eeeooceeooeeoeeeeoooeeoeeeeeeeceeeo 2.02 61‘. Bacgo agar............................... 15.00 gr. Distilled water.......................... 1000 C00. The medium was prepared on the basis of 2500 c.c., all materials being carefully weighed on an analytical balance. The MgSO4, KH2P04, ENC}, agar, and 2500 c.c. of water were placed in a 3000 c.c. pyrex flask and autoclaved at 15 pounds pressure for twenty minutes. This preheating facilitated 1 Reaction of medium after autoclaving at 15 pounds for 15:minutes was pH 6.4. 25 solution of the agar and removal of the precipitate that would occur upon subsequent heatings. The sugars were added after the medium had cooled to 60° C. at room tempera- ture. No attempt was made to filter the medium since a clear solution could be obtained by removing the supernatant fluid with a pipette without disturbing the precipitate. It was desired that the initial reaction for each series of plates at the six temperature levels studied range from pH 4.0 to pH 9.0 at intervals shown in Table Iv. The hydrogen- ion concentration was adjusted as follows. In to each of six 500 c.c. flasks was pipetted 300 c.c. of the stock medium. These flasks, tOgether with the remain- ing stock medium, were placed in a water bath maintained at 50° C. until ready for use. Ten c.c. portions of the stock TABLE IV. INDICATORS USED TO ADJUST THE REACTION OF MEDIUM. pH Desired Indicator Useful pH Range 4.0 Bromphenol Blue 3.0-4.6 5.0 Methyl Red 4.4-6.0 6.0 Chlorphenol Red 5.2-6.8 7.0 Bromthynol Blue 6.0—7.6 8.0 Cresol Red 7.2-8.8 9.0 Oleo Red B 8.6-10.2 solution were then placed in each of six test tubes and in- cubated in a beaker of water at approximately 60° C. Five- tenths c.c. of the apprOpriate sulfonephthalein or other pH indicator (Table IV) was added to each tube. The tubes were then placed in a Lamotte comparator and.N/5 HCl or N/S‘NaOH 26 added from a one c.c. pipette until the end point for each pH level was reached. The amount of normal acid or base necessary to adjust the pH of the 300 c.c. of medium in each of the six flasks was computed from these preliminary tests. These quantities were recorded. Sufficient amounts of NaOH were added to the flasks with medium in the neutral or alkaline range to compensate for the anticipated drOp in pH during sterilization. These quantities were estimated based upon previous experience. The pH of the medium from each flask was then ascertained colorimetrically1 from 10 c.c. samples. The data was recorded. Sterile dis- tilled water*was added to each flask where necessary, to equal- ize the volumes where they had been altered by adding acid or base. The content of each flask was pipetted into 20 c.c. test tubes, exactly 12 c.c. per tube. The tubes were plugged and autoclaved at 10 pounds pressure for 12 minutes. The pH was again determined and recorded. Since the pH values were approximately the ones desired to experimentally establish 'the relation of hydrogen-ion concentration to the growth of .piaporthe vexans, additional adjustment was not made. After 48 hours (tubes kept in a moist airtight container) the medium was liquefied in an Arnold sterilizer (100° C.) and 18 plates poured from the tubes at each of the six pH levels. ZHydrOgen-ion concentrations were determined immediately from the remaininngortions of the stock medium. The plates were 1 The Lamotte comparator was used. 27 inoculated with a two millimeter mycelial disc cut with a sterile glass tube from the periphery of a five day old cul- ture of the organism (incubated at 26° C.) growing on the stock medium in Petri dishes. Three plates from each pH level were incubated in DeKhotinsky Constant Temperature Tanks at the following temperatures: 2.50 C., 10° C., 26° C., 280 C., 30° C., and 34° C. The plates at 2.5° C. were placed in an ordinary laboratory refrigerator. Temperature variation in the temperature tanks was t.25° C. At the end of five days, growth was determined.by meas- uring the diameter of the colonies. If the colony was not circular, the largest diameter was measured and recorded. The final reaction for the medium in each series was as follows: pH 4.2, 5.3, 6.2, 7.0, 7.8, and 8.8. As antici- pated, a reduction occurred during autoclaving in the hydrogen- ion concentration of the alkaline media. The pH of each series before autoclaving was as follows: pH 4.2, 5.3, 6.2, 7.4, 8.4, 9.6. Slight caramelization of the medium occurred at pH 8.8 as indicated by the brown coloration. No perceptible change in reaction occurred during the period of 48 hours following preparation and before pouring the plates, nor after heating the medium to the melting point. The maximum growth of piaporthe vexans, as determined by colony measurements, occurred when the initial reaction of the medium was adjusted to pH 6.2 and the temperature maintained at 26—280 c. (Table v). This is illustrated graphically in Figures (3) and (4). 28 .oansnsmsms vow as: avian» oasaaaoosoo A+V unpack» oz AIV « .moanodoo mo noposdav.nsol ca momma monogamonmm nu muonasz H 3 mm Hm mm + I u Am.mmv Hm Am.omV Hm ”m.4mvmm + n m.m . am an on 1 i m on «m on + : “my w “mag mm Anna Hm fin.amvwm + n w.a a am On mm + n ma on em ow + 1 Anal «a Ao.omv on AomV an in.omvon + u o.a Ha mm mm mm + n ma ow «4 m4 0.4 a 2.: 3 3.5 3 as: 3 0.39 ii: 0.“ u «a ed on ow me m.v : m mm mm mm u a «o.mv o Am.amv mm Aflev ow “o.oevm< u n m.m m on «v as c an 4 on as 8 n .. R3 e 3.08 mm as a... Assam .. . «4 m «m mm on u u «n on mm on ca m.m r :3 mmamodoo mo A.asv Hoposdfin and A.o mmoawoev cuspsnomsoa suave: mo aoavomom .mzosgzmozoo STSSSH as sausage muons; S was E2 was: Hamel 35%.: B .3 3.393 .p as: , 3—.‘A, GROWTH 29 48NN 44 4O 36 32 28 . 24 _ ' 30‘ .-———-O 20 I. I6 . /\ / \ .2 - / \\ / 34° 4.2 5.3 6.2 7.0 7.8 8.8 FIGURE 3.-TNE RELATION or HYDROGEN-ION CONCENTRATION To TNE GROWTH or W yum AT VARIOUS TENPENATqus ON A SYNTHETIO NEOIUN. NEAN OIANETEII or OOLONIES AFTER FIVE OAvs. ewowTN AT IO'G. OCCURRED ONLY AT PH c.c. GROWTH 3O SONIA 45 _ 6.2 O 5.3 . ' 4O _ - 32 . ?\a.e \\ ' 25 - .ll’ \ 20 _ I, O’ M 7 l J m I 2.5“ IO“ 26' 28° 30° 34" DEGREES CENTIGRADE FIGURE 4.- THE RELATION OF TEMPERATURE To THE GROWTH OF QIAQORTg VEXANS ON A SYNTHETIC NEOIUN AT VARIOUS HYDROGEN-ION CONCENTRATIONS. MEAN DIA- METER OF OOLONIES AFTER FIVE DAYS. 31 The influence of hydrogen-ion concentration upon growth was most marked at temperatures adverse to growth. At 10° C. measurable growth occurred only at pH 6.2 and at 34° C. a marked increase was noted (Fig. 3). In genera1,growth at pH 4.2 appeared superficial and the mycelium.was very fine. The colonies were measurable, but the mycelium was barely visible unless the readings were made in bright light. A similar condition existed where the initial reaction of the medium was pH 7.8 or 8.8. At these levels the hyphae were course, sparse, and the colonies very irreg- ular. This was eSpecially true at 30° and 34° C. .At 30° 0., pH 5.3, the colony characteristics were identical to those at pH 4.2 previously discussed. However, at 26° c. the plates with an initial reaction of pH 5.3 showed a very white, lux- uriant growth. This situation also occurred at 28° C., pH 7.0. D. Effect 2; Light 3223 Growth and.Pycnidial Production. An experiment to determine the effect of light, within certain limits Of intensity, upon mycelial growth was con- ducted as follows: potato dextrose agarl, synthetic agar (p. 35), and cornmeal agar2 were used as growth media. Two hundred c.c. Of each medium was prepared in 500 c.c. flasks and the reaction adjusted to pH 6.0 with approximately N/5 H01 and N/5 NaOH . The colorimetric method (p. 25) was used to determine the re- action. The media were then autoclaved for 10 minutes at 12 pounds pressure and the reaction determined a second time. 1 120 gr. sliced potatoes, 15 gr. dextrose agar, 1000 c.c. water. 2 Dich cornmeal agar, 20 gr. per 1000 c.c. water. 32 Eight plates were poured from each flask, each plate- containing approximately 20 c.c. of agar, and inoculated with Qiaporthe‘gggggg, using inoculum and method cited in pH studies (p. ’25) . Twelve plates, four from each medium, were placed in a closed metal box, and 12, those to receive light, were Spread upon a standard drawing board (26 x 20 inches). The plates were incubated near the center Of a greenhouse (approx. 20 x 15 x 12 ft.), the drawing board resting upon the rectangular steel box. .Artificial light was not supplied. However, after 48 hours, due to low temperature and insufficient light the plates were moved to a warmer room and subjected to artificial light. The metal container was placed contiguous to the plates on the end of the drawing board which in turn was seated on a greenhouse bench. The plates were subjected to light from a 250 watt bulb suSpended 40 inches above the plates. This artificial light was supplied from midnight to six a.m. each day, the plates received daylight at other times. The temper- ature was recorded with a Taylor thermograph placed on the board. The temperature in the metal container and at the level of the plates was recorded at intervals with a mercury thermometer. The results of this experiment are summarized in Table VI. Artificial light may either stimulate or retard the growth Of Diaporthe vexans ig‘gitgg, depending upon the nature of the substrate. Under the conditions of this experiment growth was stimulated on potato dextrose agar and a synthetic agar “nth;- V 5 fi—rv- 33 medium, but retarded on cornmeal agar. Colonies produced on cornmeal agar in light were exceptionally regular. This was also true for colonies produced on potato dextrose agar in the dark. TABLE VI. THE GROWTH OF DIAPORTHE VEXANS ON VARIOUS MEDIA IN LIGHT AND DARK. DIAMETER OF COLONI IAE AFTER SEVEN DAYS. AVERAGE TEMPERATURE : 240 C., pH 6.0 65 45 Potato dextrose 65 45 (43.7)1 agar 63 (64.3) 43 02 42 21 31 21 (21.0) 25 (29) Cornmeal agar 21 31 21 C 40 24 43 (43) 25 (25-3) Synthetic agar3 27 27 2 C 1 Numbers in parentheses refer to mean diameter Of colonies. 2 Indicates contamination. 3 Formula given On page 24. Experiments conducted to note the effect of light upon the production of reproductive structure 13 ziggg were con— ducted as follows: In the first experiment, 12 tubes of corn- meal agar (reaction adjusted tO pH 6.0) were inoculated with the organism (p. 35 par. 1). Six:of these tubes were placed in a metal box (12 x 4 x 3.5 inches) containing absorbent 34 cotton, in such a way that they were in contact with the metal lid. The other six were seated on the lid directly above the inner tubes. Continuous artificial light was supplied from a shaded 100 watt bulb placed 17 inches above the tubes. A sterile thermometer was immersed in the agar of a separate tube (inoculated with the organism) and placed with the tubes exposed to light. Equipment was not available for accurately measuring the light received by each tube; however, a range of incident light values was established from tube one to six by using a‘Weston.light meter. A mirror was laid on the tubes to facilitate reading the light meter scale and the meter moved from one limit of the tubes to the other. The values were recorded. After eight days the number Of fruiting bodies in each of the 12 tubes were counted and recorded. It is demonstrated by this experiment that the formation of pycnidia is stimulated by exposure to artificial light. At the end of eight days the tubes exposed to the light con- tained numerous pycnidia (Fig. 9), while those incubated in darkness did not produce fruiting bodies. The light received by the tubes ranged from 110 foot candles for tube one to 140 for tubes four and five, to 130 for tube six. The number of pycnidia formed in each tube was as follows: TUbe N00 100OOOOOOOOOOOOOOOOOCOOOCOO 2 pycnidia 20.0.00...OOOOOOOOOCOQCOOOCZS II II II II II II II II II II II II 3.00000....eeeeeo..0000000039 4.0ooeeceoooeeeeeeeeo00.00041 500000000.00.00.0000000000041 6000.00.00...00.00.000.000032 35 In a second experiment, 500 c.c. of a synthetic agarl were prepared, and 15 c.c. poured into each Of 20 Petri dishes. The plates were inoculated with a 2 mm. loop Of Spore suSpension and incubated in a manner similar to that given on p. 32 par. 2. Ten plates were kept in total dark— ness and 10 were exposed to light. Artificial light was sup- plied On a 24 hour basis from a shaded 500 watt bulb placed 40 inches above the plates. At the end of 10 days the number of pycnidia on each plate was Observed. Hundreds of typically beaked pycnidia were produced on the 10 plates exposed to light, while no pycnidia were produced in any of the plates incubated in darkness. Perithecia did not develOp under the conditions of these experiments. LE. Carbon Utilization. To ascertain the ability of the fungus to utilize carbon from various sources when supplied separately ip 32339, the follOWing methods were employed. Varying quantities Of sugars (Table VII) were incorporated in a "sugar—free" syn- thetic medium.2 Two liters of the medium containing the 'basic compounds were prepared in a 3000 c.c. flask and auto- claved at 15 pounds for 20 minutes to allow for precipitation. I?ifty c.c. aliquots of this basic medium were pipetted into exich Of twenty-five 200 c.c. flasks. Each series of five flasks contained a carbohydrate at five different concentrations 1 Sucrose 30.80 gr., ASparagine .33 gr., KgHP04 2.610 gr., JHgSCm 1.23 gr., H20 1000 ml., Reaction adj. to H 6.2. 2 Formula given on p. 24. 36 TABLE VII. SOURCES AND QUANTITIES OF CARBOHYDRATES EMPLOYED IN PREPARATION OF MEDIA. Source . M01. I Flask Gr. per flask Gr. per Gr. Carbon Cone. No. (50 m1. aliquot) I Liter per Liter 2 a a as is .0 . . . ..3 fiffifise .09 3 .892 17.84 6.48 198.17 .12 4 1.189 23.79 8.63 .15 5 1.486 29.74 10.79 .03 6 .270 5.40 2.15 .06 7 .540 10.80 4.33 Ga1a°t°9° .09 8 .810 16.21 6.48 M.W. .12 9 1.080 21.61 8.63 180.15 .15 10 1.350 27.01 10.79 .03 11 .510 10.27 4.32 Sucrose .06 12 1.030 20.53 8.64 N.w. .09 13 1.540 30.80 12.96 349 2 .12 14 2.050 41.06 17.27 “' .15 15 2.560 51.32 21.59 .03 16 .510 10.27 4.32 Maltese .06 17 1.030 20.53 8.64 .09 18 1.540 30.80 12.96 M.w. .12 19 2.050 41.06 17.27 342.2 .15 20 2.560 51.32 21.59 .03 21 .510 10.27 4.32 LactOSQ .06 22 1.030 20.53 8.6” .09 23 1.540 30.80 12.96 A.W. .12 ‘ 24 2.050 41.06 17.27 342,2 .15 25 2.560 51.32 21.59 1Molecular Weight Alwu . .0».- can“! u.- .4:— 37 (Table VII). The medium was agitated vigorously to insure that the sugar was completely in solution. From each flask 36 c.c. was pipetted into three tubes, 12 0.0. per tube. The pipettes used to transfer the medium to test tubes were calibrated to yield 12 c.c. of medium. They were washed with boiling distilled water after the transfer from each flasl. The tubes and remaining stock media were autoclaved at 12 pounds for 10 minutes. Ten c.c. Of the remaining stock medium from each flask was used for pH determination; the colori- metric method was employed. The contents of the tubes were poured into Petri dishes and.inoculated with 2 mm. discs containing the mycelium, cut from the periphery of a seven day old culture on the stock medium. All the plates were incubated at 260 C. .A sufficient amount Of water was placed in the incubator to prevent ex- cessive loss of moisture from the medium. At the end.of five days the diameter of each colony was measured and recorded.1 Experimental results indicate that Qiaporthe vexans is able to utilize sucrose, glucose, maltose, lactose and galactose- as a source of carbon when these carbohydrates are supplied saingly in a synthetic agar medium (Table XIII). The greatest growth, as determined by colony measurements, occurred when sucrose was supplied in a .09 molar concentration (30.80 gr. jper liter). Using units of growth Obtained per gram of elemental 1 Many colonies were circular, others somewhat elliptica1.' In the latter cases the greatest Spread was recorded as the diameter. 38 carbon supplied as a basis, glucose was nearly twice as ef— ficient as other sugars. Also on this basis, the six-carbon compounds were more efficient, after five days Of growth, than the l2-carbon compounds. The most efficient utilization Of carbon Occurred when galactose, glucose, lactose, maltose, and.sucrose were supplied in .03 molar concentrations (Tables VIII-XII). This efficiency decreased progressively as the molarity of the solution increased. Poor growth was Obtained.when galactose and lactose were employed singly as the source of carbon (Fig. 5). The growth values obtained for these two sugars were nearly identical (Table XIII). At the end Of 12 days the colonies were approx- imately one-half as large as those obtained from sucrose, maltose or glucose (Fig. 6). Colony appearance, particularly zonation, varied with the sugar supplied. This is clearly illustrated in.Figure 6. IF. Nitrogen Utilization. An experiment was conducted to determine the ability of 'the organism to use nitrogen from various compounds when in- corporated separately in a "nitrogen-free" synthetic agar medium.1 Two liters of this basic medium, without sugar, were jprepared and autoclaved at 15 pounds for 20 minutes to allow for precipitation. Maltose was added after the solution had 0001ed tO approximately 60° C. The medium was not filtered. 1 Mg804 1.23 gr., KHQPO4 2.72 gr., Maltose 40.00 gr., asar 15 gr., H20 1000 c.c. 4’ I39 m.o 0.5a mm.am ma. mm.Hm o; . SS $4: NH. 8.3 mg mg: RNA 8. 8.8 mA 0.3 36 mo. 9.8. m.: m.mH mm.: no. .mm.oa dOHHQQSm soaeos Mo OmOpomH anaems sopnmo ho seam . mOHsoHoo mo Assv Ronda Rom poaamasm mo nodumup Mo nOpHH mom Rom Assv spaosc umposmae sacs "Ansonw sopnmo mo .nw Isoosoo Amaoz . omovomfl mo .na .0 com a... magma so mead mass agenda? E $033 no 289.3233. .RH mamas w.H n.5a mn.oa ma. Ho.wm H.m o.mH mo.m NH. 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Nada doaammsm adacma no omopqpm asadoa dengue no Edam ham Aaav npaouw mowqoaoo Mo Aaav umpmawad qwoa "£p3ono novaa Mom dwadmmfim nopawo mo .ya mo qoaamnp unmonop.nwaoz .0 com 94 mgoma .3 33 min gamma a 353”" um mmomopm no 55.3233 .N 33 Mo umpaa pom mmonofim no .no 41 umaaOHoo mo popoadan gums H Ao.nav wa.ma.na Am.nav ma.om.ma Am.mmv mm.mm.am Ammv mm.mm.:m Am.mmv mm.mm.om ma. Ao.uav oa.ma.ma Ao.mav ma.ma.om Am.:mv mm.mm.mm Am.mmv mm.wm.mm Ao.omv om.Hm.Hm NH. Am.oav mH.mH.mH Am.mav mH.HN.mH Am.o:v o:.a:.o: Aomv Hm.om.mm Am.:mv mm.mm.:m o0. Ao.mflv ma.mfl.pa Am.mflv mfl.ma.om a.mmv mm.wm.mm Am.wmv mm.oq.un Am.mmv mm.wm.mm mo. Am.mHV mH.uH.mH Am.mflv mH.mH.mH Am.wmv mm.mm.mm nomv Hm.mm.om HAm.HmV Hm.Hm.nm mo. omOpomA cacaodddc mmoAQSm omoufiao macaadz pawnm mo moapdnp mmfiqoaoo Mo Annapoaaaafia nflv nopoaaau was opwndmnopnwo nnooqoo “ado: zommdo ho momDom 4 m4 defiDm mDon¢5 wszD .0 com ad.mhdq fime mmahd mmwmm> fimemoden ho maaomw .HHHN finmda m.a m.mm mm.am ma. Nn.Hm m.H m.om um.ma NH. mo.a: m.H m.:m mm.ma mo. om.om m.m m.m~ so.m mo. mm.o~ H.m m.HN mm.: no. pm.oa umaamgpm auacma no omopndz aaadoa nopndo Ho swam uoadoaoo No “Hay umpaa pom cmaHQQSm no mnoapwna Mo nopaa now you Aaav avionw nopoadad game unp3on¢ nopndo mo .99 unoQQoo hdaoz omopddz mo .na .0 com admgomu Ea Bum agma a »m @8342 .8 28935234 .3" 39a 42 400m /4\ Q‘. 36 f \ 32 28 E 24 3 o n: '0 20 .. “4“ 1- <’ £M‘“- I6 “~~0~L&9_T_9§5_ ,,,,, .o__---:© l2 .. 8 . 4 _ .03 .06 .09 .l2 .ISMOL. CONCENTRATION OF SUGAR neon: 5.4m: EFFECT or vanvme QUANTITIES or suens UPON GROWTH or W m. MEAN cameras or commas AFTER FIVE DAYS A1 are. 43 Figure 6.- PhotOgraph to illustrate colony characteristics of Dia orthe vexans on a Synthetic medium containing various sugars in .12 mol. concentrations, after 12 days at 26° C. Initial reaction: pH 6.4. (A) Sucrose. (B) Maltose. (C) Glucose. (D) Lactose. (E) Galactose. Note colony zonation t pical of each sugar, i.e., very fine in (DK to very broad in (A). Growth on plate (B) is typical of that on Potato dextrose agar. 44 Fifty c.c. of the clear medium was pipetted into each of twenty-four 200 c.c. flasks containing a quantity of a nitrogenous compound (Table XIV)- The methods employed from this point to inoculation, incubation, and recording growth, were identical to the procedures outlined for the carbohydrate work (pp. 35-37) except for the following: The pH of the tubed media was altered during autoclaving. The method employed to adjust the reaction to the desired level was as follows. Methyl Red, in .6 c.c. quantities, was added to each tube which re- quired readjustment. All the tubes were maintained at approx- imately 60° C. in a water bath. A tube containing 12 c.c. of stock medium was adjusted to pH 5.0 using Methyl Red and the Lamotte comparator. Quantities of N/5 HCl and.N/51NaOH were added aseptically to each tube until the color change was equal to that of the tube previously adjusted to pH 5.0. To each tube not receiving Methyl Red .6 c.c. of sterile dis- tilled water was added. Although growth was recorded after five days, the plates remained in the incubator for a period of 16 days. The results are summarized as follows. When ammonium sulphate, potassium nitrate, separagine, and peptone were em- ployed singly as the source of nitrogen, asparagine gave the greatest growth (Table XV). Asparagine proved also to be the most efficient source of nitrOgen when millimeters of colony growth per gram of nitrogen supplied, were used as a basis for comparison (Table XV). Peptone was the least effic- ient source of nitrOgen, although very good growth occurred ‘45 .>x capes com H ammonia no «Hana Benson mo «no.5 250930 mo sauna mum.m o.<~ 0N.a «N moo.~ 0. ma 0. MN mam.~ o.ea b. «N H¢H.¢H . oaaaama 0.3...” 0.0." m. 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