acre-1' ENETIATMN mo newsman m VACUNEUM ggaymmgggg g WITH REFERENCE To THE ammsm 0?- Asmmmm 222.25%: 'E‘hes‘ts far We Degme 3% P31. 0.. MECMEGAH STATE Cir‘l_.§.,53a‘3;i 39322“: $12216? Mafififiade 7..» C1 fl I! } '5 ~13 t. c: s F2 -\ «S. ‘KJ Gun-u. Inf-dvhduhaf. Jr. .71 _ . r . . , :1 ‘1’... I1. . a O. . l. . Thisictocerdfgthat tlie ‘ , thesis entitled » . " "ROOT INITIATION AND DEVELOPMENT ‘ IN VACCINIUM CORYMBOSUM L. WITH ‘ REFERENCE TO THE INFLUENCE OF ASSOCIATED FUNGI." . presented by ‘ John Peter Mahlstede has been accepted towards fulfillment of the requirements for Eb. D. degree infionthnlture ‘ Major professor Date I ' 5b a 1*- ‘-’-_ 0-169 . .I- I- ‘l u‘ I l ‘____,_‘....,_!__:_ _ -II _ ROOT INITIATION AND DEVELOPMENT IN VACCINIUM CORWWBOSUM L, WITH REFERENCE TO THE INFLUENCE OF ASSOCIATED FUNGI By John Peter Mghlstede A Thesis Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1950 ‘. ‘ 1 ACKNOWLEDGENENTS The author is deeply indebted to Dr. Donald P. Watson of the Department of Horticulture, for valuable suggestions and direction throughout the conduction of these studies. He is grateful for the respected counsel of Professors H. B. Tukey and F. L. O'Rourke, and for the criticisms and suggestions of Dr. Everett Beneke and Mr. Harold Davidson, all of Michigan State College. An expression of deep gratitude is directed to Alberta Mahlstede for her constant encouragement and support throughout the period of this investigation. TAFLE OF CONTENTS Page I}:TRODUCTIO}IOOOOOOIOOOOOOOOOOOOOOIOOOOOOOO0000......0.. 1 DISCUSSION OF PERTINENT LITERATURE..................... 3 Mycorrhizal....................................... 3 Physiological..................................... 7 Anatomical........................................ 9 EXPERIMENTAL PROCEDURE................................. 15 Materials......................................... 15 Cultural techniques for the first experiment...... 17 Cultural techniques for the second experiment..... 19 Mycological techniques............................ 22 Hist010gical techniques........................... 2h EXPERIMENTAL RESULTS AND EXPIANA'IIONS.................. 27 Anatomy of the stem of Vaccinium corymbosum £..... 27 Origin and development of root primordium......... 28 Description of associated fungi................... 32 Relation between isolated fungi and root growth... 3h DISCUSSION OF RESUDTS.................................. ul Negative relationship between presence of fungi and number of root initials..................... Effect of associated fungi on root initiation and development............. ooooooooo coo-50.0.00 LlJ-l- Fungi aSsociated with endotrophic hyphal activity. MS Mechanical inhibition of root projection.......... R6 Phyaiological..."..O....‘C....................... 1+8 Superiority of root production by proximal contrasted to distal stem parta................. #9 SUMRYOOCOOOOOOOOO0.0000000COOOOCOIOO0.0.0.00.0.0.0... 52 BIBI‘IOGRAPHYOOOOOOOO000......0.0....OOOOOOOOOOOOOOOOOOO Sh m1...3..1.11 I s O I p o - o v t . a n a v u . O O u I I u a O . 0 C . O O . o O O O O I O Q C C O nun. .I. 1,...wr.,..u.ulflh .32» .u.‘ NTRODUCTION Many plants are known to exist in close association with fungi, an interrelation between the fungus and the roots of the higher plants that is commonly called a mycorrhiza. When the plant or any part thereof contains hyphal fundaments, the relationship is either symbiotic or parasitic. This relationShip complicates the nutrition.of these plants and introduces speculation concerning the role of the fungus element. Friesleben (193h) indicates that the influence of the mycornhizal fungus on the growth of the associated plant species is the result of the inactivation, destruction, or absorpt- ion of repressive substances in the rooting medium. Stimulation of root activity may be induced by the release of natural auxins from.the individual hypha to the surrounding medium (Rayner, 1938). More recently many investigations concerned with the vegetative propagation of plants have been based upon the stim- ulation of adventitious roots by plant auxins. The stage of maturity of most plant parts governs partially the quantity of auxin present, and consequently influences the ability to regenerate missing parts. To clearly interpret adventitious root initiation it becomes necessary to understand the anatomical phases of 2 development, whether one approaches this phenomenon.from the point of view of the influence of the auxin or the influence of fungal activity. The present problem is designed to determine the anatomical aspects of root initiation and development in Vaccinium.corymbosum,g, with the presence or absence of associated fungal forms. DISCUSSION OF PERTINENT LITERATURE Mycorrhizal: Association between fungi and higher plants is not a recent concept. Numerous references have been made (Osborn, 1909; Weiss, 190k; Halket, 1930) to the presence of mycorrhizae in plants belonging to the Palaeozoic era. It was not until the third century B.C. that any mention of the occurrence of fungi with the roots of higher plants did exist. Theophrastus in the third century B.C. made note of the occurrence of fungi with the roots of oak and other trees ({elley, 1950). In 193A Asai recorded the presence of mycorrhizae in one hundred and.ten out of one hundred and thirty- four families of higher plants. He assumed that these fungi were parasitic deriving elaborated food from the plants. Frank described die morOphological nature of ectotrOphic mycorrhizae. He explained the association as one of symbiosis whereby the plant's supply of water and mineral salts is increased as a result of the presence of the fungus; the fungus in turn used carbohy- drates that were manufactured and stored in the plant. Stahl (1900) supported Frank's observations and advanced the theory that the mycorrhizal fungi associated.with the roots of higher plants growing in a soil rich in organic matter enable the plants to acquire food through the increased area afforded by the numerous hyphae. h He believed that this increase in surface area increases the supply of water and.mineral salts instead of reducing them by competition from separate micro-organisms inherent in the medium. In contrast to the opinion of‘Frank and Stahl, McDougall (l9lh) concluded that the fungus was a parasite. This parasitic relationship is regarded by Burges (1936) as an example of a controlled attack because the activity of the fungus is curbed by the reaction of the host. Melin demonstrated in 1925, by pure culture techniques, that there was no significant difference between absorption by an infected and non-infected nnot. To support the theory that endotrophic mycorrhizae liberate and make available soil bound elements, Ternetz (1907) isolated species of Ehgmg_from.Vaccinium and demonstrated that this genus possesses a limited capability for fixing nitrOgen. Rayner (1929) using aseptic innoculation reported that Phoma radicis oxycocci and vaccinii are capable of fixing nitrogen and are stimulated by the presence of organic matter in the soil. Knudsen (1929) using species of Calluna and Freisleben (l93u) using species of‘Vaccinium have reported that seedlings could normally develOp without the associated fungi. Since the fungus has been located in seeds by Rayner, the seedling may contain fungus soon after germination. Waksman (1916) maintains that in S soils high in organic matter the microflora are composed of many fungi in no way connected to the mycorrhizal complex. By attacking the humus of the soil they liberate critical nutrients required for the growth of higher plants. He states that these micro-organisms may syn- thesize fats, waxes, hemicelluloses or proteins which are absorbed to the humus in the soil. Jahn (193h) used the term "petotrOphic mycorrhiza" to describe this concept and found £333; and Penicillium to be the most common genera indigenous to this soil. The general classification of the fungal component of the mycorrhizal complex consists of four general types; the ectotrophic, the endotrOphic, the ectendotro- phic, and the pseudomycorrhiza. The ectotrophic mycorrhiza refers to a definite morphological organ which consists of a slow growing short root and fungi that are regularly arranged (Hatch and Doak, 1933). The endotrophic mycorrhiza refers to the association of the plant part and the fungus that is located inside the plant. Fossil records indicate their presence in the pre-historic period of civilization (Butler, 1938). Rayner (1929) reported the presence of endotrophic mycorrhizae in the genus Vaccinium as an annual occurrence in the plant. She observed that the mycelium in the stem is extremely fine, is associated with the 6 cell wall, and is inter and intra-cellularly located. Addoms and Mounce (1931) working with the cranberry, Vaccinium macrocarpon reported that all pasts of the stem contained equal quantities of the fungus. In the young stem the fungus was noted in all regions except the epidermis. McDougall (19lh) has observed in Quercus and 5223 that the mycorrhizae appear during the summer in small numbers, increase substantially during the fall and winter, and die during the spring. The endotrophic fungus of Vaccinium macrocarpon is found more easily near the tips of the rapidly growing stolons and is most conspicious in parenchymatous cells of the pith and cortex (Addoms mid Mounce, 1931). They noted dark masses in the ray elements which they believed presented progressive phases in the digestion of the hyphal straids. Rayner (1929) suggests that mycorrhiza formation in the genus Vaccinium is an annual activity influenced by the existing soil conditions. This is in agreement with the findings of McDougall for Quercus and £333. Dufrenoy (1917) and Doak (1928) have also reported the presence of endrotrophic forms in the various tissue systems of Vaccinium. The ectendotrophic relationship, a combination of ectotrophic and endotrophic forms, is described by Hatch and Doak (1933) as being found consistently in a limited number of species. 7 Pseudomycorrhizae resemble the mycorrhizal complex in appearance. Fungal activity is not beneficial to the plant associate. Invasion of roots by pathogenic fungi result in a decreased absorptive ability which subsequently results in the death of the plant in severe cases of infection. Physiological: The production of roots on a cutting is dependent upon the formation and maintenance of root initials from the tissues situated distally on the severed stem. Many external factors have been shown to influence the production of adventitious roots on stem cuttings. Hitchcock (1928) working with a variety of plant species observed death of tissue accompanied by browning on the lower end of cuttings that rooted poorly in an acid medium. Partial or complete neutralization of the media tended to permit the deve10pment of callus and subsequent rooting without any apparent injury to the stem base. Cuttings of some species produced roots in acid peat moss provid- ing it was abnormally wet. It has been suggested that this may be attributed to the higher intercellular oxygen content in the cambium regions (Sledge, 1930). Although cuttings taken in April and September produced roots, the style of cutting had no consistent influence on the root- ing response of Vaccinium corymbosumig, (tare, 1930). He found that the number of roots produced was directly related to an increase in stem diameter. Chadwick (1932) found that by collecting hardwood cuttings in late winter or early spring to reduce the storage period, he could avoid much bacterial and fungal infection as well as prevent dessication of tissues. O'Rourke (19hh) observed that vegetative wood of Vaccinium corymbosum L. rooted more readily than flowering wood. He also obtained a higher percentage of rooting ability on basal than on terminal stem pieces, with intermediate rooting ability on the medial pieces. There is no evidence to Show that the location of the basal cut influences the rapidity or quantity of adventitious roots. Chadwick (1930) working with deciduous softwood.cuttings found more roots when the basal cut was one half inch.below the node, while O'Rourke (1950) found with hardwood stem cuttings of the highbush blueberry, that the position of the basal cut made no apparent difference in the resultant number or type of adventitious roots. The relationship between adventitious root product- ion and age of the tissue has been known for many years. Gardner (1929) reported that in general, hardwood apple cuttings taken from the outer perifery of young trees produced the greatest number of adventitious roots. In 1932, Hitchcock concluded that the rooting response of various greenwood cuttings was dependent upon the age of the material used. In.hardwaod stem cuttings of Mngg domestica Bock two distinct growth phases were observed to be in effect (Stoutemyer, 1937). The young or juvenile condition was favored by repeated cell division, but was not anatomically different from older mature wood. Cuttings made from the juvenile wood produced a greater quantity of roots, and this was concluded to be caused by a physiological process rather than a direct anatomical manifestation. Van Overbeck (l9h5) attributed the lack of adventitious root formation in a white variety of Hibiscus to a deficiency of natural aixin produced in the leaves. Anatomical: An attempt to learn the origin of adventitious roots in vascular plants has been made by -many investigators. Van Tieghem and Douliot (1888) reported that young adventitious roots in stems arise endOgenously from the pericycle. They observed that the semi-mature parts of stems may produce roots from the phloem parenchyma and in mature wood adventitious roots originate from the cambium. They noted that lateral roots of Hypericum pyramidatum arose in the pericyclic region of the root. The root initial was first observed by the radial elongation of several bilateral pericyclic cells, with subsequent tangential divisions that projected the primoridium radially. The endodermis, cortex, and epidermis was ruptured by rapid cell divisions and elong- ation of the primordium. The initiation and development of lateral roots on terminal roots was concluded to be 10 synonymous with initiation and development of advent- itious roots on young stems, rhizomes, and stolons (Van Tieghem and Douliot). Van der Lek (1925) has reported that in older stems adventitious roots arise close to medullary rays and in association with secondary tissues. Apparently it is impossible to make general- izations concerning the exact tissue involved in the initiation of adventitious roots in stem cuttings. The origin varies with the stage of maturity of the Shoot at the time of adventitious root development (Priestley and Swingle 1929). They have described the Change of the arrangement of tissue with age. With the development of the vascular cambium the ray cells in the cambium region are compressed and adventitious roots arise in association with the cambium. They reported that as growth continues the root initials arise in a group of undifferentiated air-free cells adjacent to the cambium. Connard and Zimmerman (1931) observed that in young stem cuttings of Portulaca oleracea L. adventitious roots generally arose in rays adjacent to the primary bundles, in comparison to their appearance in smaller connecting bundles where in older stems they were independent of any previously formed vascular tissues. Adventitious roots in stem cuttings of Coleus blumei L. were produced between U18 fibrovascular bundles originating in one to several adjacent cells of the pericycle (Carlson, 1929). ll Carlson (1933) found that in semi-woody canes of the Dorothy Perkins and American Pillar roses the origin of adventitious roots was centered in single or in small groups of parenchyma tissue of the vascular cylinder at the base of the shoots. Van der Lek (1925) reported that young branches of Salig sp. and.Populus sp. produced adventitious roots in connection with the cambium at the end of a medullary ray. In young stem cuttings of Lonicera jgponica adventitious preformed root initials were produced from a group of cells extending from.the cambium to the pericycle in contrast to adventitious roots induced by vegetative propagation that developed from the cambium layer (Sandison, 1932). Wolfe (l93h) reported that adventitious roots in Cotoneaster Dammeri were produced as a result of the activity of one of the two groups of parenchyma cells in the divided bud gap. Priestley'and Swingle (1929) have Observed that the development of adventitious roots on hardwood cuttings is directly related to the activity of the buds. They attribute it to "conditions governing meristematic activity in the neighborhood of the proximal wound and that delicate internal balance which converts this activity from the production of ordinary new vascular tissue to the organization of root growing points.” This relation- ship hinges on the cambium which is the medium between the activity of the bud and.the initiation of new root 12 primordia. They further noted that the resumption in cambial activity was governed by many factors, one of which was the displacement of air by sap in the inter- cellular spaces in tissues near the region of root initiation. The production of adventitious roots in relation to the production of callus tissue at the base of woody cutt- ings has been investigated with a variety of vegetatively propagated plants. Knight (1926) and Zimmerman (1925) concluded that callus formation and root production were two individual processes. Knight observed that, although abundant callus formation accompanied prolific advent- itious root formation the two processes were "collateral manifestations of internal conditions" and that "the relation between them is very susceptible to external conditions." Although hardwood cuttings of Prunus domestica, 3. cerasus, and Malus domestica produced abundant callus at the basal end of the cutting very few roots developed (Zimmerman, 1925). The formation of callus on stem cuttings of Hibiscus Rosa-sinensis L, and Hgyig brasiliensis resulted from the activity of the medullary ray system and was not related.to the activity of the cambium (Sharples and Gunnery, 1933). Adventitious rpots may be produced from callus tissue under Optimum environmental conditions (Swingle, 1929). However, Knight (1926) has reported that callus formation is favored 13 by high water content of the soil in contrast to root formation which is favored by a greater supply of oxygen. The development of root primordia is believed to be re- tarded in callus tissue and consequently root emergence is limited to older, suberized callus tissues (Zimmerman 1925). In 1926, Swarbrick reported that wounds made on growing Sycamore, Rhododendron, plum and apple plants exhibited a seasonal variation in "wound gum" formation. He noted that fu>m wounds made between the months of May and October partial or complete blocking of the vessels occurred as contrasted to the November to April period when no blocking took place. The normal sequence of wound cork formation resulted from the alteration of a cellulose surface to cutin or suberin followed by the accumulation of sap along the cut surface and subsequent stimulation of activity of a phellogen in the parenchyma region (Priestly and Woffenden, 1922). Priestly and Swingle (1929) recognized the significance of callus and cork formation when they considered vegetative propagation by cuttings. The healing or blocking of these exposed surfaces prevented the influx of saprophytic and parasitic fungi and possible disintegration of contiguous stem tissue. The blocking of exposed tissues also reduced loss of moisture from the cut at the base of the cutting. There has been considerable emphasis placed upon stem anatomy and its relation to the capability of a woody 11+ cutting to produce adventitious roots. Val der Lek (1925) concluded that the production of adventitious roots (morphological roots) near the base of the stem cuttings is restricted to certain morphological locations which were determined by the arrangement and type of tissues of the cutting. In several species of willows he found preformed root primordia, to which he gave the name "root germs". In the course of his study, van der Lek (1925) made the observation that, in general, root primordia arise from a definite group of cells at a definite location in the stem but are not discernible from surrounding tissue at the time the cutting is made. He did not name or locate these regions. 15 EXPERIMENTAL PROCEDURE Materials: For the first experiment, one year old stem cuttings of Vaccinium corymbosum.§,, variety Jersey were collected in a dormant condition in Harch of 19L9 from a four year old planting growing in a peat bOg north of Lansing, Michigan. Some material used in.the second experiment was obtained from an established planting at the experiment station, South Haven, Michigan. This material was collected in the greenwood stage of maturity during the month of August, 1950. Other material was of the same type used in.the first experiment. For the initial trial a modified solar frame, divided into four sections was constructed similarly to a convent- ional cold frame (Figs. 1 and 2). Two inch lumber was used for the frame; the overall dimensions being six feet in length, six feet in width and two feet eight inches in depth. The frame was divided into four separate compart- ments which were lined with four cutting trays. These inserts (two feet eleven inches square) were constructed of six inch boards, covered with one-quarter inch hardware cloth stapled to the underside, and placed on braces nailed to the inside of each compartment twelve inches above the floor of the frame. The second experiment required a special propagating frame that would allow frequent sampling and complete aeration as well as insure aseptic conditions. For this HOUR! I. 5 ~H\ ’ l "L rw fugue I ( .440 sand I f l T l 0 W E I | w . « *1» -, ..—,1 w— at. AA 594“ m m .N { K I 1 ' (“501 “IL A 4FJ‘L w FIGURE 8. - -- a A ,, - 72‘— ,_ P; w‘.‘ E J— FIG.1. End elevation of propagation frame. FIG.2. Plan view of propagation frame. J: W ____-___A__36' _ _ __ _ __ _ 16 purpose a "Mahlstede" propagation chamber was designed and constructed. After a trial period in which Coleus blumei proved the success of the frame, ten additional boxes were constructed for the second experiment. Each box consisted of three parts: the outer shell, the insert, and the cover (Figs. 3 and S). The outside measurements of the white pine shell were twenty inches in length, twelve and one-half inches in width, and twelve inches in depth. On one side of the box a board four inches in width was hinged to the shell to allow aeration of the structure. In the top of one end partition an isosbceles -triang1e, (six by five by five inches) was cut out, grooved along the inner base and fitted to cover the removed section. The six inch deep insert was constructed of one-half inch white pine sheeting (eighteen and one- half by eleven and one-half inches). To the underside of this insert was stapled one-quarter inch hardware cloth, and it was covered with a one-half inch layer of rock wool held in place by cheese-cloth netting. To facilitate accessibility, a small triangular section was removed from one end partition to coincide with the opening in the shell. The cover (twenty-one and three-quarter inches by thirteen and one-half inches by three inches) was sealed with a single pane of glass. The entire structure was sealed with plastic wood and coated with aluminum oil paint. FIG.3. Propagation chamber, end view. FIG.4. Hardwood cuttings of Vaccinium corymbcsufl L- variety "Jersey", showing callus (left) and beginning of roots (right) after 45 days. PT.~. C: Dnnwfinr‘n 9' ‘g nv‘ nhnmkr.\n Pvann 4- 1'4 n1.- 17 Cultural techniques for the first experiment: To insure an accurate comparison throughout these invest- igations, cuttings were of uniform diameter, eadh shoot containing not less than four cuttings. The two diStal parts were designated as terminal, and the two proximal parts as basal, cuttings. When a shoot could be divided into more cuttings, the medial section of the whip was discarded. Cuttings were four inches in length, their terminal horizontal cuts approximately one-eighth inch above, and the basal diagonal cuts immediately below, the bud. All cuttings were placed in an air-tight container into which had been introduced 0.01 percent propylene oxide (Hansen and Snyder, lth). The cuttings were allowed to remain for a period of two hours after which they were soaked in a solution of 7.2 percent slaked lime for a period of ten minutes. The cuttings were rinsed in two changes of sterile distilled water. Two hundred and forty terminal, and two hundred and forty basal, cuttings were placed in a complex of ten fungi, which had been isolated previously from.similar stem tissue and root systems during the growing season. This group is later referred to as the treated material. An equal number of cuttings was soaked in a sterile chamber in sterilized distilled water for a period of ten minutes. This group is later referred to as the untreated material. Each insert in the modified solar frame which had been filled 18 previously with peat moss and sterilized in a steam chamber (180°F) for two hours was transferred under aseptic conditions to a transfer room. Each lot of cuttings was placed twenty per row with one hundred and twenty cuttings of each type confined to each insert. Cuttings were set on the ninth of July 19h9 in the conventional manner in a vertical position with the apex of each cutting approximately one inch above the surface of the medium. This procedure was repeated for both treated and untreated plots, each insert sub- sequently being transferred to one of the four prepared culture chambers of the modified solar frame which had been sterilized with a ten percent solution of potassium permanganate. All plots were covered with standard sash. During the months of July and August, the sash was covered with an emulsion of lime, water, and oil to reduce the temperature within the frame. In addition, four inches of water was applied daily to the bottom of each section to maintain a high relative humidity and to eliminate Opening for the purpose of adding water to the cuttings during the rooting sequence. The mean temperature main- tained during the 88 day propagation period was 82.50F above the cuttings and 80°F in the rooting medium. Peat moss used for the rooting medium gradually became less acid, changing throughout the period of rooting from a pH of 0 to 5.0, the untreated plots consistently having h. a slightly higher reading than the treated plots (plus 0.5). 19 The sash was allowed to remain on.the frames for a period of one and one-half months after which all plots were watered with sterilized distilled water and covered with screen netting. Samples of five cuttings from.sach of the four lots were taken at ten day intervals after the first fifteen days had elapsed (Fig. h). Cultural techniques for the second experiment: The procedure adopted for the first experiment was repeated using instead of the mixture of ten fungi, a single fungus for each trial. Ten fungi were isolated from the mixture and one each of these was applied to individual lots of cuttings. Seven hundred cuttings made from the medial section of one year old whips (70 per glass dish) were soaked for ten.hours in one of the following nine inocula: Qphiostoma pilifera, Echinobotrium laeve, fgnicillium gpinulosum, Epicoccum isolate _1_, Epicoccnm isolate _2_, Fusarium isolate ;, Fusarium isolate g, Streptomyces sp., and Chaetcmium globosum. Each insert was filled with peat moss and the entire structure was sterilized for a period of one and one-half hours at 180°F in a stemn sterilizer. They were then transferred under aseptic conditions to the transfer room. Each lot of cuttings was removed individually into an aseptic room.Where it was inserted in the previously prepared propagation chamber (Fig. 5). The cuttings were set ten rows per box, seven per row with the apex of each cutting approximately one inch above the surface of the “I “I: ‘. A“ o 33"" a“- .o— ‘ ..a~. ‘ ‘ 4 .-6 I q I u u‘n .‘ .- b ...v -- 53,. . , r- .1 5| ‘ b. . -"-.' ‘ ... 's4 . r. , I "V N‘ .g, j 20 medium. This procedure was repeated for the eight re- maining treated lots and for the untreated sample. In order to control the temperature and light inten- sity in the prOpagation chambers, a walk-in refrigerator was designed to maintain sterile conditions throughout the experiment (Fig. 6). The floor of the refrigerator was re-covered with tar paper felt and the entire structure washed with a ten percent solution of potassium permanganate. Ten twin-tube banks of fluorescent lights were arranged to maintain a light intensity of 300 ft. candles above each individual growing chamber and the temperature was regulated to 65°F. In addition to the main storage room, a lead-in de-contamination closet was constructed of tar paper felt attached to a frame, and furnished with a small hinged door. This closet was coated with an oil base aluminum paint and fitted with a prOpolyne vaporizer system to de-contaminate the Operator before entering the refrigerator. Two weeks after the experiment was initiated, the refrigeration failed to operate and as a result of the heat radiated by the light system, the temperature rose to 139°F. The entire procedure was then repeated using a second lot of hardwood cuttings. Three weeks after this trial had commenced, a second malfunction in equipment developed and the plant material was destroyed. The following method was then adopted to substitute for the preceding procedure. Greenwood cuttings were the FIG.6. PrOpagation chamber under fluorescent lights in controlled temperature chamber. 21 only commercial stock available in August of 1950. To minimize the number of replications, each whip of the current seasons growth was divided into four sections, using only the two medial and discarding the terminal and basal parts. Cuttings were made four inches in length and leaves removed at all but the two terminal nodes. The terminal horizontal cut was approximately one-eighth inch above, and the basal diagonal out immediately below, the bud. The cuttings were surface sterilized as before, soaked for a period of ten hours in separate fungal solution cultures covering the basal two inches of each cutting. 0n the 15th of August 1950, each lot was set seven per row in ten rows in its designated propagation chamber. The propagation cham- bers were then transferred under aseptic conditions to individual compartments of a divided cold frame. This frame was nine feet in length, six feet in width, and two feet eight inches in depth. The frame was further divided into ten compartments, each completely sealed from the other. The frames were covered with.three glass hot bed sash. During the month of August, the sash was covered with one-quarter inch mesh camouflage netting to minimize the temperature within the frame. The entire structure was coated on the inten.or with aluminum paint and soaked with a twenty percent solution of potassium permanganate. After transferring all of 22 the chambers, the entire system was again surface sterilized with a ten percent solution of potassium permanganate and sealed. The frames remained sealed until the 13th or October 1950 (60 days), at which time they were Opened and the individual propagation chambers transferred to a greenhouse bench illuminated by two banks of twin-tube fluorescent lamps. Samples of five cuttings from each Of the ten lots were taken 60 and 105 days after the cuttings were set. MycolOgical techniqpes: In order to secure fungi associated most commonly with Vaccinium corymbosum £3 under Michigan conditions, weekly isolations from stan and root tissues were made during the period between August 19h8 and March l9h9 preceding the first experiment. Stem and root material from which isolations were to be made was collected from an established bog planting near Michigan State College. Euds were removed from the stems to eliminate all possibility of fungal contamination by spores and hyphae that might be enveloped within the overlapping bud scale system. General laboratory procedures to avoid contamination were followed. Dissection and transfer instruments were sterilized in 70% alcohol, heated, and immersed in 70% alcohol when not in use. A 7.2% solution of slaked lime followed by two rinses of sterilized distilled water was used to sterilize the stem and root parts. This was followed by a dip of 70% alcohol and rapid flaming. One, 23 two, and three year old Stems as well as primary, secondary, and teritiary roots were cut into thin slices approximately 500 microns in thickness to facilitate growth of the endotropic mycelia. The six different kinds of thin slices were re-sterilized.before being placed on blueberry agar (infusion.from.500 grams of blueberry stem.tissue and 15 grans of agar). Five divisions were made on each agar plate to afford a place for one root or stem disc. After a two week period, single spores or hyphal tips were isolated from the various colonies that developed from each.disc. These fungi were then cultured on potato-dextrose agar (in- fusion from potatoes 200 grams, Bacto-dextrose 20 grams, agar 15 grans) or Czapek's agar (water 1000 ml., NaNOB 3 grams, KZHPOu 1 gram, MgSOu.7 H20 .5 gram, FeSOh.7H20 .01 gram, sucrose 30 grams, agar 15 grams), for iden- tification. To identify non-sporulating types, it was necessary to culture some of the isolates on slide-culture (Riddel 1950). The first experiment incorporated the use of a com- plex of nine fungi previously isolated and selected for use throughout these trials. This complex was cultured on 500 ml. of potato-dextrose agar in 1000 ml. Erlenmyer flasks. Five hundred ml. of sterilized distilled water was then added to the flasks after the colonies had develoPed fully. The fungus colonies and agar were thoroughly mixed with sterilized distilled water and 2h transferred to sterilized square glass refrigerator dishes to which the cuttings were subsequently added for a ten minute soaking period. The techniques for the second experiment were modified to facilitate the addition of the fungi to the cuttings. Following isolation of the fungi, individual hyphal fragments from each culture were transferred to separate solutions of Fries' hedia Which was made up to the following specifications: Ammonium Tartrate 10.0 grams Ammonium Nitrate 2.0 grams Potassium Dihydrogen Phosphate 2.0 grams Sodium Chloride 0.2 gram Calcium Chloride (Anhydrous)0.2 gram Magnesium Chloride 1.0 gram Sucrose 30.0 grams in 2000 ml. of sterilized distilled water. Seventy cuttings were soaked in sterilized glass refrigerator dishes containing 100 ml. of the culture solution of the specific fungus to be tested. Sufficient sterilized distilled water to submerge all cuttings was added and the entire system rotated to insure complete mixing. Histological techniques: All cuttings were sectioned and prepared for microscopic observation in a similar manner. All cuttings were transferred from the individual propagation chambers to a moist atmosphere in square glass refrigerator dishes and stored for less than two days before sectioning. The basal slant cut 25 was divided into three regions: (1) the tip to the center of the pith (2) the center of the pith to the summit of the cut, and.(3) remaining part of the stem. The distal one and one-quarter inch section of each fresh stem part was then removed and inserted in a vertical position in the clamp holder of a Bausch and Lomb sliding microtome. Transverse sections were cut approximately 35 microns in thickness. The ranainder of the stem was then cut longitudinally at the same thick- ness. Four representative transverse sections from.sach of the regions one, two, and three in addition to eight longitudinal sections were selected and transferred for eight hours to a three percent solution of acetic acid containing orseillin BB (Strassburger, 1923). After rinsing in forty percent alcohol, the.materia1 was stained for ten minutes in a solution of analine blue in three percent acetic acid. Washing and dehydrating with alcohol was followed by clearing in clove oil and xylol. Permanent mounting in Canada balsam was done in such a manner as to orient sections for future identifica- tion. The most desirable staining was characterized by a light purple color. Lignin, suberin, nuclei, and cyto- plaan stained red (orseillin BB); cellulose stained blue (analine blue); hypha stained shades of violet and blue (orseillin BBiand analine blue). water soaked and dead tissue appeared yellow or brown. It was necessary to 26 modify the portion of the stem selected for sectioning in those cuttings of the second experiment that portrayed basal browning in excess of one and one-quarter inches from the base. In this case a one and one-quarter inch part was cut in such a way as to include a three quarter inch region of each of the brown and normal regions. Resultant transverse sections were classified as to origin following the previous technique. Serial sections of several stems were made in order to ascertain the region of root initiation. For'this purpose distal three-quarter inch stem.pieces were softened with sodium hydroxide and hydrofluoric acid (Hyland, 19hl), and imbedded in the usual paraffin.method. Transverse sections were cut on a rotary microtome and permanent slides prepared by dehydrating, staining with Haiden- heins haemotoxylin and safranin and mounting in Canada balsam. 27 EXPERIMENTAL RESULTS AND EXPLANATIONS Anatomy of the Stem of Vaccinium corymbosum L.: (Fig. 7) The cuticular layer (a) on the surface of one year old stems of Vaccinium corymbosum.§. covers a single epidermal layer (b) of cells. Many other plants from a moist habitat are frequently found to have a thin cuticle, however, on all plants examined, the cuticle is thick. It is approximately the same depth as the subtending epidermal cells. The epidermis consists of a single row of closely packed isodiametric cells, which toward the perifery of the stem have blunt, saw-toothed apices. The cuticle although entire is interrupted regularly by stomata (a) (Fig. 8) extending into the cortical region. The guard cells are thin walled, but the wall is thicker on the side close to the aperture. All of the cortical cells are isodiometric. They are smaller and more closely packed toward the outer edge of the cortical region. The elliptical to globose cortical cells subtending the stomatal mechanism are grouped more closely beneath the cavity and are somewhat smaller than those throughout most of the cortex. The cortical region varies from five to twelve cells in depth and deliniates a solid tissue system between the epidermis and air canals. Most cortical cells contain many chloroplasts. Centripetal to the cort- ical layer are large air ducts, usually two in a group, bilaterally located, and ranging from.three to four / . . Q; . ,. . B I ° "’2 " - . . K. -‘ I -. a .\ i Q - '", l .. . o ' " Q ."o . '0' . - , ‘0 . , s - . O 0 3 .° 0 0 'p ‘ .‘*.. '0 . t . . . 0 ‘0 . . . ' . .' O O .. C o D Q C - . - . .' O ....G"&' .‘a I O ‘ \ ’ 6 fl . a . ' ‘ ‘ .. . \ ' ‘ ‘ o“'o ‘Qa . ~‘< Qz<.MBH>HBo< Admmwm 0HmmomBOsz om mqmda 11.1 DISCUSSION OF RESULTS Negative relationship between thegpresence of fungi and the number of root initials: It is not pr0posed to suggest that all of’the results are sufficiently com- prehensive to suggest alterations in the practices of prepagation of this plant. The number of roots produced was not sufficiently high to be considered typical for every season. In general, the addition of fungi to the propagation medium did not increase the percentage of rooted cuttings. In all cases the untreated cuttings contained large numbers of endotrophic hyphal filaments. This would indicate that an appreciable amount of fungus was in the stem at the time the cuttings were removed from the plant. The mycelium.was located adjacent to the vascular cambium in the outer xylem vessels in contrast to its location in all xylem, phloem, pith, and cortical regions of the greenwood stem. One probable explanation of the location of hyphae is the different amount of water available in various stem tissues at different stages of maturity. In the rapidly growing greenwood stem, the pith, xylem, phloem and cortical tissue contain large quantities of cellulose and bound water that would make a good medium for mycelial growth. As a result of the greater similarity of the chemical composition of all tissue at a young stage, the selectivity of a region suitable for fungal growth 12 would not be as great as in the older stem. As the stem matured, more lignified cells of the older xylem would not make as desirable a medium for-the growth of fungi as would the young xylem vessels. The movement of large quantities of water in the older vessels might impede fungus growth. Less free water would be absorbed by the lignified, than by the cellulose, vessels and consequently the medium.would not be as good for fungus growth. The quantity of carbohydrate in all tissues of the green stem might also improve growing conditions fir the fungi. The results indicate that where there is a medium number of hyphal filaments in the stem.tissue, the greatest number of roots develop. After ninety-five days all of the untreated hardwood cuttings display the best rooting ability and contain concentrations of hyphae in the medium range. Where hyphal activity is either heavy or light after ninety-five days for the treated lots, the number of rooted cuttings is considerably reduced. When the quantity of hyphae is light, the sub- sequent percent of rooted cuttings is not noticeably increased, and on the contrary, when the concentration of endotrophic hyphae is in excess there is no noticeable increase in.the percent of rooted cuttings. This would appear to mean that a definite optimum quantity of endotrophic mycelia conducive to rooting might be estab- lished. 1+3 The greenwood cutting trial produced only a sparse number of rooted cuttings after 105 days in the rooting medium. This may have been caused by low'temperatures in Michigan in September and October. The decrease in temperature would reduce the growth rate of both the cutting and the fungus. The various fungi apparently responded differently to this change in temperature. Epicoccum isolate at increased in concentration with the elapse of time reaching its optimum.between 60 and 105 days. It may not have been given sufficient time to stimulate the root initials. Typical hyphal filaments observed in the longitudinal stem sections could not be identified or distinguished from any fungus that developed on agar culture media. The hyphae were much branched, septate, and brownish in color in regions where the death of the tissues occurred. In live tissue the fungus mycelium stained shades of violet. In comparison.the longitudinal sections of the hardwood cuttings revealed a typically non-branched, single, non-septate hypha which stained a deep blue. These hyphae were observed passing through.the pits in.the cell walls of the young xylem vessels. It is suggested that the stimulation of root initiation in hardwood cuttings is brought about by the digestion of these hyphal strands and that these hyphae represent a source of available nutrient substances, which at a definite concentration stimulate the production of root m initials. This observation is in agreement with McDougall (l9lh) who reported.that the mycorrhizae of guercus and £223 appear during the summer in small numbers, increase during the fall and.winter and die during the spring. However in the greenwood cuttings the hyphae are active and non fused in.which state the substances assimilated by them is available in a mnall quantity. Effect of associated.fungi on root initiation and development: The cuttings that did not produce roots or callus at the end of 95 and 105 days exhibited a browning of the lower end of the stem in contact with the medium. It is thought that this is caused in part by the influx of saprophytic fungi into the non-living xylem elements. Adjacent ray and xylem parenchyma, as a result of the presence of the fungi form tyloses and subsequently secrete a wound gum.deposit in the infected tissues. The formation of gum.renders complete the functions of the secreting cell in the nutritional process of the cutting. The cells are dead but are physiologically importarm to the plant. They prevent the healthy wound tissue from absorbing water and air. This hindrance to the normal oxygen supply decreases the infection.of the hyphal filaments, and these saprOphytic forms generally require a certain amount of oxygen in order to carry on their life processes. LLS The breakdown of the cuticle, epidermis, and cortex is another probable function of associated fungi. The first action of some of these fungi is to dissolve the middle lamella, with subsequent direct penetration into the surrounding tissue. As the fungus progresses from the epidermis via the stomatal Opening, through the cortex, its progress is frequently halted at the pericylic fiber region. Developing root initials are then aided in their lateral projection.by the weaking of the pericyclic fiber region and the separation of the cells centripital to it. Fig. 15 illustrates the quantity of adventitious roots emerging from.this characteristic brown area at the proximal end of the cuttings. Fungi associated with endotrophic hyphal activity: The identity of individual fungal supplements could not be determined in the longitudinal sections of material used in the second experiment. The lack of production of fruiting bodies within the "host" tissue coupled.with the modification of the hyphal strands in.the tissues of the stem further led to the complication of such a procedure. In the first and second experiment, perithecia were produced very shortly after the cuttings were set in the sterilized rooting medium. They appeared at the surface of the basal slant cut, and where the epidermis was ruptured by the knife at the time the cutting was made. It would be reasonable to conclude that the FIG.15.Roots developing through dead brown cortex and epidermis. h6 endotrophic hyphae present in the stem material at the time of collection produced these fruiting bodies at the exposed ends of the severed xylem elements. This fungus was identified to be gpbiostoma pilifera (Fries) Wint., which is commonly associated with the)"bluing" or "red rot” of western yellow pine. The fungus could have been introduced from the surrounding plantations of Scotch pine, Pinus sylvestris, or from the lumber in the prOpagation frames in spite of the cuttings being main- tained under what was thought to be sterile conditions. Intermediate trials in the laboratory have shown that perithecia were produced in all cases, no matter what particular method of surface sterilization was utilized. From.these data it is suggested that hardwood and green- wood cutting material used in these trials contained gphiostoma pilifera (Fries) tint.....inberent in the stem. This is further substantiated by the isolation and identification of Graphium from stem tissue during the initial isolation trials. Graphium sp. has been iden- tified.as the imperfect stage for several of the Qphiostoma .species. Mechanical inhibition.of root projection: The ring of pericyclic fibers and heavy cuticle cementing the outer tips of the epidermal cells are suggested as two characteristic mechanical obstacles to the projectioncaf the root. The photomicrograph of a transverse section through the developing root (Fig. 9) shows the bending of in the initial before it reaches the cortex. 1t is believed that the pericyclic bending is produced as a result of the inability of the developing initial to break through the continuous ring of thick-walled pericyclic fibers. There is no evidence to indicate that the initial at this point dissolves the surrounding tissue in its trans- verse movement toward the perifery of the stem. It is suggested therefore, that the pericyclic fiber region offers a mechanical barrier to the root initial, which is forced to change its course. There is evidence of contraction of the cell mass in the cortical region of the new root as it passes through the pericyclic fibers. The root attains a greater diameter'both inside and out- side of these fibers. A curvature of the developing root directly beneath the epidermis at as great an angle as at 90° is shown in Fig. 10. The radial projection of the developing root is halted as a result of the mechanical resistance offered by the thick cuticle and.densely packed cortical parenchyma. Only in severe cases does the developing primordium make a right angle bend and dis- continue further activity. In less severe cases specimens have been observed Where a root bent slightly and sub- sequently forced its way through the cortex, epidermis and cuticle at some point far removed from the loci of a point directly radial to its origin. This would cause a root to be malformed. It might be injured mechanically. 118 It might not break the outer epidermal and.cuticular layer. When.the cuticle or epidermis does break, it may happen at a point of mechanical weakness or injury in the cuticle. Frequently when.the epidermis is dead, degeneration of the tissue may precede root emergence. Either pericyclic fiber or cuticular restriction may be one cause of the low percentage of rooting on the stem cuttings of this species. If this is the primary cause of obstruction, rooting percentages could be raised by using a weak acid or solvent to destroy or dissolve the cuticle, the epidermal layer, and even the pericyclic fibers providing no damage was done to the phloem or cambium.region where the initials originate. Physiological inhibition of root prgjection: The bending and apparent retardation of elongation of the young roots (Figs. 9 and 10) may be attributed to an insufficient supply of nutrients, water, oxygen, or elaborated food at this stage in its development. It may be a result of a combination of mechanical and physiological causes. Because root development was less and less rapid in the greenwood cuttings, mechanical obstruction is a more likely cause of root inhibition. There would be a greater supply of food in.the phys- iologically more active greenwood,than,in the less active hardwood stems. Anatomical differences as a result of age would further complicate this type of conclusion. #9 There was a great difference in the amount of lateral growth on each lot of hardwood cuttings. Treated basal cuttings for example averaged h2.l cm. and untreated basal cuttings averaged 29.9 cm. in length after ninety- five days. It would be expected that these cuttings would produce roots in relation to the amount of leaf area but this was not true. It did not prove to be true for the terminal cuttings and it was concluded that there was no relation between.the leaf area available for syn- thesizing food and the quantity of roots. The lack of consistency between rooting ability and opportunity for additional supply of elaborated food tends to discount the theory of inhibition of root projection as a result of insufficient food. Better evidence might be obtained through tests of rooting in a media where sugar was supplied. Superiority of root production by proximal contrasted to distil stem parts: The number of cuttings that pro- duced roots after ninety-five days was conspicuously low for all hardwood lots of cuttings. This is in accord with the results obtained by were (1930) working with the southern blueberry. He reported eight percent root- ing for cuttings made during the month of July. The low percentage of rooted cuttings is unfortunate since the experiment was designed to determine What influence associated fungal forms might have on the production of SO roots. The experiment was terminated after a sufficient number of cuttings had produced roots, and it was possible to establish the influence of the fungus on rooting. The average number of rooted cuttings is therefore small because of the interruption at a time when all cuttings had not yet produced roots. As has been previously noted (O'Rourke, l9hh) hard- wood cuttings taken from.the basal portion.of one-year old whips of Vaccinium.corymbosum.§. produced a greater quantity of adventitious roots in comparison to those cuttings made from terminal stem pieces. The untreated basal lot of cuttings of the first experiment did not sub- stantiate these results. However the number of cuttings sampled is insufficient to indicate that there exists a significant difference. One explanation for the greater number of roots produced per rooted cutting may be the increased activity of the endotrophic fungi, which render mineral elements and elaborated food.products available to the cutting. Ericaceous plants grow frequently in humus or acid peat soils, which are notably deficient in nitrates. It is recognized that certain mycorrhizal fungi can readily assimilate ammonium and organic nitrogen com- pounds, which are not available otherwise to the cutting. As has been.previously suggested by Rayner (1927) for Calluna, the possible role of the mycorrhizal element in.the cuttings of Vaccinium corymbosum Q. is one of 51 availability and the absorption of organic food reserves, which are unavailable to the cutting in alkaline soils. The most important function of the endotrophic hyphal filaments is probably the increased absorptive surface afforded by the mycelium located in the xylem vessels of the stem and extending into the rooting medium. It must be pointed out however, that there is no adequate explanation for the means of absorption.of substances by the hyphal filaments. Although the root-hair hypothesis for the absorption of substances exists, there is in- sufficient evidence to indicate that hyphal strands are able to act in this capacity. The possible formation of excretions by the endotrophic hyphae may also result in.the higher percentage of roots per rooted cutting taken from the proximal end of one-year old whips. Subsequent diffusion of these substances along the medullary ray system to the region of root initiation might stimulate adventitious root formation. 1. 52 SUMMARY Greenwood and hardwood.cuttings of Vaccinium corymbosum 2. were rooted under aseptic conditions to study the influence on rooting of a fungal com- plex as well as nine separate associated fungi. Detailed description is given of the methods used for these experiments including the techniques of innoc- ulation and control of environmental influences. Stem anatomy is discussed in.relation.to the plant part used for propagation purposes. Roots were found to originate in the cambial and protophloem.regions and project through the callus or through the outer stele, cortex and epidermis. Descriptions are given of the mechanical inhibitflan of root projection by the lignified pericyclic fibers and closely packed epidermal cells cemented together by a thick cuticle on the outside of'the stem. Lack of elaborated food, water and oxygen at the time of early root development is suggested as a further possible reason for inhibition of root projection through.the pericycle and the epidermal layer. The nine fungi associated with the stems and roots of this species are identified and described. No relationship is established between their presence and the amount of root deve10pment. 53 Associated fungi were found to break down the outer tissues of the stem in preparation.for*rpot emergence. With a moderate amount of endotrophic fungal activity, there was more rooting than with a higher or lower amount of activity. Sh BIBLIOGRAPHY Addoms, R. M. and Mounce, F. C. Notes on the Nutrient Requirements and the Histology of the Cranberry (V. Macrocarpon) with Special Reference to mycorrhiza. Plant Physiology 6:653-668 Illus., 1931. Asai, T. Uber das Vorkommen und die Bedeutung der Wurzelpilze in der Landpflanzen. Jap. J. Botany 7: 107. 1934. Burges, Alan. On.the Significance of Mychorrhizae. New Phytologist 35:117-131, 1936. Butler, E. J. The Occurrences and Systematic Position of the Vesicular - Arbuscular Type of Mychorrhizal Fungi. Brit. Mycol. Soc. 22:27h-30l, 1938. Carlson, Margery C. Microchemical Studies of Booting and Non Rooting Cuttings. Bot. Gaz. 87:6h-81, 1929. Carlson, M. C. Comparative Anatomical Studies of Dorothy Perkins and Am. Pillar Rose, 1. Anatomy of Canes 2. Origin and Development of the Adventitious Roots in Cuttings. Contr. Boyce Thompson Inst. 53313-330, 1933. Chadwick, L. C. Factors Influencing the Rooting of Deciduous Hardwood Cuttings. Proc. A.S.H.S. 28: ASS-59. 1932. 10. 11. 12. 13. 15. 16. 55 Chadwick, L. C. The Influence of the Position of the Basal Cut on the Rooting and Arrangement of Roots on Decidious Soft Wood Cuttings. Proc. A.S.H.S. 27=u87-u9u. 1930. Connard, Mary H. and Zimmerman, P. w. The Origin 5: Adventitious Roots in Cuttings of Portulaca Oleracea L. Contr. Boyce Thompson Inst, 3: 337-h6, 1931. Doak, K. D. The Mycorrhizal Fungus of Vaccinium (Abst). PhytopatholOgy 18:1u8, 1928. Dufrenoy, J. The Endotrophic Myoarrhiza of Ericaceae. New Phytol. 16:222-228, 1917. Frank, A. B. Uber die Auf Warzelsymbioes Beruhande Ernahrung Gewisser Baume durch unteriridische Pilze. Ber. d. dent. Bot. Ges. 3:128-1h5, 1885. Freisleben, R. Zia-Frage der MykotrOphie in dur Gattung Vacciniume. Jahrb. Wiss. Bot. 80:h21-h56, 193A. Gardner, F. E. The Relationship between Tree Age and the Rooting of Cuttings. Proc. A.S.H.S. 26: lOl-th, 1929. Gregory, L. E. and van.0verbeek, J. An Analysis of the Process of Root Formation on Cuttings of a Difficult-to-Root Hibiscus Variety. Proc. A.S.H.S. hézh27-k33. Halket, M. The Rootlets of Amyelon Radicans Will; their Anatomy, Apices, and Endophytic Fungus. Ann. Bot. hh386S-905, 1930. 17. 18. 19. 20. 21. 22. 23. 25. 26. 56 Hansen, H. H. and.Snyder, W. C. Gaseous Sterilization of Biological Materials for Use as Culture Media, PhytOpath. 37(#5)=369-71, 19u7. Hatch, A. B. and Doak, K. D. Mycorrhizal and Other Features of the Root System of Pinus. Jour. Arnold Arboretum lhz85-89, 1933. Hitchock, A. E. Effect of Peat Moss and Sand on Rooting Response of Cuttings. Bot. Gas. 83 #2, 1928. Hitchcock, A. E. Relation of Rooting Response to Age of Tissue at Base of Greenwood Cuttings, Contr. Boyce Thompson Inst. h:85, 1932. Howard, N. O. The Control of Sap-stain, Mold, and Incipient Decay in Greenwood with Special Reference to Vehicle Stock. U.S.D.A. Bul. #1037:s-7, 1922. Hyland, F. The Preparation of Stem Sections of WOOdy Herbarium Specimens, Stain Technology Volume 16, April 19u1. Jahn, E. Die Peritrophe Mycorrhiza. Ber. Deutsch. Bot. Ges. 52:h63, l93h. Kelley, A. P. Mycotrophy in Plants, Chronica Botanica Co. Waltham Mass. 1950. Knight, R. C. The Propagation of Fruit Trees by Stem Cuttings. 1. Observations on the Factors Governing the Rooting of Hardwood Cuttings. Jour. Pom. and Hort. Sc. 5:2u8-266, 1926. Knudsen, L. Seed Gennination and Growth of Calluna vulgaris. New Phytolog. 28:369-376. 1929. 27. 28. 29. 30. 31. 32. 33. 3h. 35. 36. S7 Lek, H. A. A. Vander. Root Developments in Woody Cuttings. Tex. H. Vernnan and Joneu. Wageningen, 1925. McDougall, W. B. On the Mycorrhizae of Forest Trees. Am. Journ. Bot. 1:51-7h, 191k. 4 Meyen, J. Ulber das Herauswachsen Parasitisher Geuachse aus den wurzeln anderer Pflanzen. Flora 12:49-63, 1829. O'Rourke, F. L. Wood Type and Original Position on Shoot with Reference to Rooting of Hardwood Cuttings of Blueberry. Proc. A.S.H.S. 85:195-197, 19hh. O'Rourke, F. L. Unpublished data, 1950. Osborn, T. G. B. The Lateral Roots of Amyelon Radicans and their Mycorrhiza. Ann. Bot. 23:603-11, 1909.. Priestley, J. H. and Swingle, C. F. Vegetative PrOpagation from the Standpoint of Plant Anatomy, U. s. D. A. Tech. Bul. 151, 99 pp illust., 1929. Priestley, J. H. and Woffenden, L. M. Physi010gica1 Studies in Plant Anatomy V Causal Factors in Cork Formation, New Phytologist 21:252-268, 1922. Rayner, M. C. Mycorrhiza: an Account of Non-pathOgenic Infection by Fungi in Vascular Plants and Bryophytes New Phytol-Reprint #15, 2H6 pp. (Abstract from abstract) 1927. Rayner, M. C. The Biology of Fungus Infection in the Genus Vaccinum, Ann. of Bot. h3z55-70, 1929. 37. 38. 39- to. 1+5. #6. 58 Rayner, M. C. The Use of Soil or Human Inocula in Nurseries and Plantations. Empire Forestry Journal 17:236-2u3, 1938. Riddle, R. W. Permanent Stained Myc010gical Separations Obtained by Slide Culture. Mycologia u2'(#2): 265-270, 1950. Sandison, S. Rooting of Cuttings of Lonicera jgponica New Phyt010gist 33:211-217. 193A. Schrenk, H. von The "Bluing" and the "Red Rot" of the Western Yellow Pine with Special Reference to the Black Hills Forest Reserve. U.S.D.A. Bur. Plant Industry Bul. 36:t0p-1u pl. 1903. Sharples, A. and Gunnery, H. Callus Formation in “ Hibiscus Rosa-ginensis L. and Hayes brasiliensis. Plant Propagation, 1933. Sledge, W. Rooting of Wood Cuttings Considered from the Standpoint of Anatomy. Journ. Pom. and Hort. Sc. 8:12-23, 1930. Stahl, E. Der Sinn der Mycorrhizen bildung. Jb. Wiss. Bot. 3&3539. 1900. Stoutemyer, V. T. Regeneration in Various Types of Apple Wood. Iowa Res. Bul. 220, 1937. Strassburger, E. Das botanische Praktikum. Jena. 7th Ed. pp 392 and 767, 1923. Swarbrick, T. The Healing of Wounds in Woody Stems. Jour. Pom. and Hort. Sc. 5:98-11h, 1926. in. hB. 50. 51. 52. S3. 59 Swingle, C. F. A Physiological Study of Rooting and Callusing in Apple and Willow. Journ. Ag. Res. 39 #2:81-128, 1929. Ternetz, C. Ueber die Assimilation des Atmospherischen Stickstoffes durch Pilze. Jahrb. Wiss. Bot. hh:353- u08, 1907. Van Tieghem, P. and Douliot, 5. Reseaches Comparative of the Origin of the Endogenous Members of Plants (Vascular), Ann. Sci. Bot. 7.8:1-660, 1888. Waksman, 5. A. Soil Fungi and their Activities. Soil Sc. 2:103-156, 1916. Ware,_L. M. Propagation Studies with Southern Blueberry. Mississippi Ag. Expt. Sta. Bul. #280, Aug., 1930. Weiss, F. E. A Mycorrhiza from.the Loner Coal Measures. Ann. Bot. 18: 255-265, 190k. Wolfe, Florence. Origin of Adventitious Roots in Cotoneaster dammeri. Bot. Gaz. 95:686-9h, 193k. Zimmerman, P. W. Vegetative Plant Propagation with Special Reference to Cuttings. Proc. Amer. Soc. Hort. Sc. 22:223-28, 1925. 1 11. . A l f a 1. .q- 4 ‘I‘ I I‘! .\I ( I!“ ..<,I|u. 0| lll'rv‘riif it... 305.). llllllllllllfl I l I ll ll