sammelbericht. localization of the endophyte in mycotrophic organs

Zeitschrift fur Allg. Yikrobiologie

Sammelbericht

4 2 1964 165-180

Localization of the endophyte in mycotrophic organs

ARTHUR PIERSON KELLEY~)

(Eingegangen ana 14 .5 . 63) I

That fungi live with underground or creeping plant organs has, after much hesitation by more than one genwation of plant scientists, been formally admitted as truth. It is conceded that fungus-roots or mycorrhizae exist ; also, mycorrhizomes and mycothalli. Conservative botanists fear to go much further; and even studcnts of mycotrophy are reluctant t o admit cognizance of the exact processes in the fungal exchange. Few of them realize what know- ledge of the mycotrophic process lies buried in the literature; and the object of this paper is to uncover some of that evidence and bring it publicly forward. A key t o the problem is localization of the endophyte; which, once admitted, leads rapidly to other considerations.

I . Localization of endophytic infection in Hpgatics Authors who have written on mycothalli assert tha t the fungus of the mycothallus is

restricted in its invasion. This restriction is to the ventral portion of the thallus; and there are certain portions never invaded by the fungus; viz., the chlorenchyma, the central strand and the apical region.

Let us consider briefly what each of these authors says about mycothalli: The hepatic Conocephalus (Pegatella) conica is infected through rhizoids, said BEAUVERIE (19O2), and hyphae fill internal cells in a limited zone of central tissue, where vesicles are formed. Likewise in Nonocleo Porsteri, a New Zealand hepatio (according to CAVERS, 1903), the invading mycelium is limited to a sharply defined zone consisting of 2-4 layers of cells filled with branching fungal hyphae: This zone is confined to the thicker median portion of the thallus, and extends to within a short distance of the growing point.

CAVERS also found infection in Lunularia, which is the most studied of myrothallic hepn- tics. Thus, RIDLER (1923), found that Lunularia contained a fungus which occurs in a single strand of cells along the thickened median portion or midrib towards the ventral surface of the thallus. The occurrence of the fungus is not, however. constant; for it undergoes partial digestion by the hepatic, ending in formation of arbuscles and sporangioles, its growth and consequent distribution in the thallus being thus restricted. So, too, did NICOLAS (1924) find infection restricted in male thalli of Luaularia, the fungus being confined t o a band which runs the length of the mid-vein, parallel to the lower surface and removed from i t by several layers of immune cells rich in starch. I n other male thalli, the is fungus localized in cells throughout the thallus, the two sorts of infertion being possibly due t o two different fungi. Later (1929), NICOLAS reported that the endophyte occurs only in the vegetative part of the gametophyte. ATJRET (1930) likewise reported limitation of the endophyte in Lunularia: The gametophyte contains a fungus which is confined t o a definite zone below the assimilating tissue. It occurs also in the rhieoids and amphigastra but does not penetrate the gemmae cups and archegonia. And as NICOLAS reported for male mycothalli, EMBERGER (1924) stated that in female and in sporogenous plants, and in sterile thalli, the mycothallic hyphae occupy in general a large band which is separated from the lower surface of the thallus. Inconstance of infection and its physiological neutrality negate the hypothesis tha t infection is necessary to formation of sexual organs: Neither does it seem t o be a parasitic relation, for the infected thalli behave like the uninfected. ,,I think with NICOLAS", said EMBERGER, ,,that the association is simply accidental; and that localization of infection is conditioned by differences of osmotic pressure."

l ) Mt. JACKSON, Va., USA

166 ARTHUR PIERSON KELLEY

RIDLER, whose work on Lunularia is cited above, also worked on Pellia: In this hepatic the fungus was found t o occur in a definite zone along a thickened median portion towards the ventral surface of the thallus, and in the rhizoids. The fungus occurs in proximity to antheridia and archegonia. Later (1923), RIDLER described arbuscles and sporangioles from Pellia, and said that this plant seems to exercise some control over the fungus, keeping it from becoming parasitic. It was noted that formation of arbuscles limits growth of the fungus.

In Aneura, the endophyte was seen (by DENIS, 1919) to form coils in the ventral portion of the thallus; and regularly in the rhizoids.

Seuwdiella, a tuberous hepatic of southern India, normally harbors a mycothallic fungus which penetrates trough rhieoids into tissues of the young plant. Progress of hyphal growth in the tissues is checked by the active meristematic cells; while the bulb is always immune. These facts the author (CHALAUD, 1932) found in remarkable accord with BERNARD'S dis- coveries about orchids.

Finally, we may note tha t M. STMIL (1942), working with numerous species of hepatics, found that in simply constructed sorts the mycelium is confinrd to under layers of cells in the thallus, the under epidermis being mostly free. I n Lunularia, green tissues are shunned by the fungus. Artuscles and vesicles occur in all infected layers, and digestion also. In hepatics possessing conducting strands, the fungus is restricted a t a little distance, and the strand and assimilating tissues are never invaded. There is also a separation into a host- and digestion-zone, the latter limited to storage tissue.

Summary: All workers on mycothalli of hepatics say with one voice tha t the endophyte is localized in its distribution within the thallus. The mycelium is confined t o certain tissues, while other tissues are never invaded; viz., green tissues, conducting tissues, and meristems. Why are these certain tissues never invaded ? Several investigators reply tha t the hrpatic is able to limit the fungus in its growth; while two of them say localization of infection is conditioned by differences of osmotic pressure.

L i t e r a t u r e c i t a t i o n s AURET, T. B. 1930, Observations on the reproduction and fungal endophytism of LurLulaYia

BEAUVERIE, J., 1902. &ude d'uneH6patique h thalle habite par un champignon filamentaux.

CAVERS, Fr., 1903. On saprophytism and mycorrhiza in Hepaticae. New Phytol. 2: 30-35. CHALUAD, G., 1932. Mycorhizes e t tuberisation chez Sewardiella titberifera Kashyap. Ann.

DENIS, M., 1919. Sur quelques thalles d' Aneura depourvus de chlorophglls. Compt. Acad.

EMBERGER, L., 1924. A propos du Lunularia cruciata (L.) D u x e t des formations mycorhi-

NICOLAS, G. 1924. Formations mycorhiziques dans une hepatique A thalle (Lunulnriu

-, 1929. Observations sur un endophyte de Lunularia cruciata (L.) DUM. Rev. Bryol.

RIDLER, W., 1922. The fungus in Pellia epiphylla (L.) CORDA. Ann. of Bot. 36: 193-207.

-, 1923. The fungus present in Lunularia cruciata (L.) Dunf. Trans. British Mycol. SOC. 9:

RTAHL, M., 1942. Die Mykorrhiza der Lebermoose mit besonderer Beriickeichtigung der thallosen Formrn. Diss. Wiirzburg.

I I . Localization of the endophyte in Pteridophytes Of the Fern Alliance, the Ophioglossaceae and the Lycopods have been most studied for

mycotrophism. I n Ophioglossum vulgatuna L., in the outer layers of cortex, occur masses of yellowish or brownish ,,protoplasm", in a behaviour resembling that of orchids: so said VAN TIEQHEM (1870/71). This condition was also found by BOWER (1902) in 0. simplex. He said: ,,A peripheral band [of cortex]. . contains the ,grumose' masses characteristic of mycorrllizae : notwithstanding the only partial recovery of the section from drying, there is no room for doubt that the root had been mycorrhizal, evidences of the fungus being seen in some 4-5 layers of outer cortex. The characters of the cortex are very closley matched [with those of 0. pendulum], including the mycorrhizal band and the layer (probaly exodermis) with the thickened outer wall."

cruciata (L.) Dumortier. Trans. Brit, Mycol. SOC. 16, 163-176. 8 fig.

Compt. rend. Acad. Sci., Paris. 134: 616-618.

Bryol. 5: 1-16.

Sci., Paris. 168: 64-66.

ziques des hkpatiques. Bull. d. 1. SOC. d. Sci. nat. d. Maroc. 4: 211-215.

vulgaris MICHELI). Compt. rend. Acad. Sci., Paris. 178: 228- 230.

2, 35-40.

A1 0, 37. 483-487, 1923.

83-92.

Localizatio of the endophyte 167

The gametophyte (prothallus) of 0. pendulum was studied by LANG (1902) : He found that the tissue of the young prothallus is throughout parenchymatous, the cells of the lower half containing an endophytic fungus while those of the upper portion are free from it. Throug- hout further growth of the prothallus, the endophyte is confined to internal tissues, the outer cells being only traversed by infecting hyphae. In the older region of a branch, the fungus occupies all the cells save a superficial zone of one or two layers. Thus the mycothallus of Ophioglossum is esentially like that of an hepatic, the endophyte being limited to a special zone in the ventral region of the thallus.

Of Botrychium, GREVILLIUS (1895) investigated 12 species, finding that an endophytic fungus always occurs in the roots, even in B. ternatum where it was not found by KUHN. In cells of the outer cortex were clumped hyphae, and there were intracellular vesicles. Hyphae without coils were found near the tip while later a mantle was formed (that is, within the cortex) which consisted of in:

B. lanceolatum- of even 7 layers of cells; B. matricaefolium and B. simplex- of 4-7 layers B. Girginianum - of 4 layers; B. boreale - of 2-5 layers; B. obliquum - fungal structures wanting in older roots. An endophyte was found in the prothallia as well as the root of Botrychium by BAAS-

BECEING (1921). The fungus is abse7t from tissues inside the endodermis of the roots and where camlcial activity is marked the fungus is invariably absent. At a definite distance from the epidermis the fungus branched copiously inside the cells, forming arbuscles which disintegrate. The so-called vesicles occurred, thin-walled and apparently containing osmotic products.

The protallia of Botrychium were studied by JEFFREY (1898), who found an endophyte entering by way of the rhizoids: After penetrating 2 or 3 layers of cells, the hyphae coil or expand into thin-walled vesicles which are often so abundant as to fill the cells with a bo- tryose mass. In older prothallia the symbiont is shrunken and appears digested. Similar phenomena were described by BRUCHMANN (1906) : Infection may take place directly through surface of the prothallus, and an endophyte was present in every specimen that was examined, living in all the inner, radially formed, older portions. Starch, said BRUCH- MA“, is present only in meristem and in cells about the reproductive organs, while in those cells which have no starch the hyphae are filled with oil and protein. BRUCHMANN found an endophyte in the sporophyte of Botryclchium also; and the outer cortex of the root was filled with hyphae which, however, never penetrate the epidermis, root-tip nor central cylinder

Turning next to Psilotum, we find (according to BERN~TSKY, 1899) that it contains an endophyte which produces sporangia-like bodies that he called sporangioids : he considered them to be diseased sporangia. Another writer, DARNELLSMITH (1917), said that the sporel- ings of Psilotum are penetrated by an endotrophic fungus after a comparatively few cell divisions; and soon almost a11 of the ceIls of the prothallus are filled with a skein of hyphae. Infection occurs near the growing point and in the sex organs, but not in the egg-cell.

In another genus of the Psilotaceae, Tmesipteris, we find also an endotrophic fungus, said DANGEAD (1891) : The fungus forms great coils in cortical cells of the my;orrhizo ne, I n the prothallus of this plant (LAWSON, 1917), many cells are filled with an endophytic fungus, the older prothallia having practically all the cells exeept the superficial ones so filled; and the hyphae finally disintegrate. LAWSON thought there was no localization of infection, yet he said the fungus was never found in superficial cells nor in the archegonia. He also stated that prothallia of Psilotum are much like those of Tmesipteris.

The Marattiaceae are also mpcotrophic (KUHN, 1889): Infection is directly through the epidermis and hyphae grow in the inner cortex. Gummy yellow ma88es are produced in 2-3 layers of cells. Russow (1872) had also found that “two of three layers of inner cortex stand out from the others because of the content of their cells, and aggregate mass which is tinged faintly with yellow.”

In Cyathea, JANSE (1897) found pelotes and sporangioles in 3 - 4 layers of cortex; while in Opkioderma pendulum, branched hyphcte and sporangioles were found in the third layer of cortex only.

In prothalliit of the fern Cheiropleura (NAKAI, 1933), a genus usually included in the Polv- pdiaceae, fungal hyphae were found entering by way of brown rhizoids and filling the median part of the prothallus, branching and coiling and forming a “nutritive layer”.

Infection, in the form of coiled hyphae, was noted by VAN TIEGHEM (1870/71) in large cells of the inner cortex of Osmunda regalis.

Continuing with the Lycopods, we find GOEBEL (1887) stating that the lower non-meristematic portion of the prothallus of Lycopodium innundatum was always, without exception,


inhabited by a fungus; and that the fungus was limited t o one or two outer cell layers, seldom penetrating deeper; forming a zone which is separated from the exterior by several cell layers. The infected cells do not contain starch but drops of oil. I n American species of Lycopodium, APESSARD (1922) found the fungus spreading t o within 2- 3 cell-layers of the meristem, the mycelium coiling in the lower part of the prothallus, the 4th layer from the outside; and hyphae do not enter the palisade. In New Zealand lycopods, HOLLOWAY (1920) found the fungus growing forward with the prothallus, occupying a zone between the epidermis and the central conducting cells.

Fossil lycopods tell the same story: WEISS (1904) found a fossil root which he considered lycopodiaceous, and referred to the form genus Rhizonium of CORDA. Infection was for the most part in the inner cortex where hyphal swellings were found; and the author also described a specialization of the cortex into Pilzwirtzellen and Verdauungszellen.

Summary I n all cases reported, Pteridophytes show a localization of the endophyte. In the gameto-

phyte, the prothallus resembles that of the hepatic in having the endophyte limited to a region in the ventral part of the prothallus; and never growing into meristem, archegonium, or conducting strand. I n the sporophyte, the endophyte is confined to certain layers of cells in the cortex, never invading areas of cambial activity or meristem, nor the central cylinder. At a definite distance from the epidermis the fungus begins t o branch and form those structures which will be digested in phagocytosis: this is usually in the inner cortex but the exact location is within a certain layer of cells in the cortex, which is specific for each species of Pteridophyte. Likewise, in the fossil forms there is the same specific localization of infection, the cortex alone harboring the endophyte and the fungus breaking doivn in the inner cortex a t a set distance from the stele.

L i t e r a t u r e c i t a t i o n s BAAS-BECKING, L. G. M., 1921. The origin of the vascular structure in the genus Botrychium,

BERNATSKY, J., 1899. Adatok az endotroph mykorhizhk ismeretkhez. Termesx. Fiizet.

BOWER, F. O., 1904. Ophioglossum simplex RIDLEY. Ann. of Bot. 18, 205-216. BRUCHMANN, H., 1904. uber das Prothallium und die Keimpflanze yon Ophioglossum

DANGEARD, P. A. C., 1891. Note sur les mycorhizes endotrophique. Le Botaniste. 2,

DARNELLSMITH, G. P., 1917 (1918). The gametophyte of Psilotum. Trans. of the Roy.

GOEBEL. K.. 1887. Uber Prothallium aund Keimpflanzen von Lvcovodiuin innundatum.

with notes on the general anatomy. Rec. d . Trav. bot. Nekrland. 18,333-372.

22, 88-110.

vulgatum L. Bot. Zeit. 62, 227-248.

223-228.

SOC. of Edinburgh. 62 (L), 79-92. Y -

Bot. Zeit.'46, 161-167; 177-199. GREVILLIUS, A. Y., 1895. uber Mykorrhizen bei der Gattung Botrychium nebst einigen

Bemerkungen iiber das Auftreten von Wurzelsprossen bei B. virginianuna SWARTZ. Flora. so, 445-453.

Part 11'. Trans. &; Proc. of the New Zealand Inst. 62, 193-239.

Jard. bot. d. Buitenzorg. 14, 53-201.

University Library.

HOLLOWAY, J. E., 1920. Studies in the New Zealand species of the genus Lycopodium.

JANSE, J. M., 1897. Les endophytes radicaux de quelques plantes Javanaises. Ann. d.

JEFFREY, E. C., 1898. Tho gametophyte of Botrychium virginiannm. 32 pp. Toronto, The

BURN. R.. 1881. Untersuchungen iiber die Anatomie der Harattiaceen und anderer GefaB- kryptogamen. Flora. 72, 46-504.

LANC. W. H.. 1902. On the urothallia of Ovhioqlossum vendulum and Helminthostaehvs - I

zeylanica. Ann. of Bot. 16, 23-56.

SOC. Edinburgh. 62 (l), 93-114.

grifolia. Bot. Mag. Tokyo. 47, 1 - 5.

1'Akad. d. 80. d. Petersbourg. 19, 107-11s.

LAWSON, A. A., 1917 (1918). The gametophyte generation of the Psilotaceae. Trans. Roy.

NAKAI, T., 1933. An observation on the gametophyte of Cheiropleuria bicuspa var. inte-

Russow, 1872. Verglrichende Untersuchungen der Leitbiindelkryptogamen. Mem. d.

Localization of the endophyte 169

SPESSARD, E. A., 1922. Prothallia of Lycopodium in America. 11. Rot. Gaz. 74, 392-413. v. TIEQHEM, P., 1870/71. Recherches sur la symbtrie de structure des plantes vasculaires.

WEISS, F. E., 1904. A mycorhiza from the Lower Coal Measures. Ann. of Bot. 18,255 -265. WEST, C., 1917. On Stigeosporum muraltiacearum and the mycorhisa of the Mnrattiaceae.

Ann. d. Sci. nat. Rot. Fime. ser. 13, 1-314.

Ann. of Bot. 31, 77 - 99.

I I I . LocaZization of the endophyte in Gymnosperms I n the fossil Cordaites, known as Amyelon radicans, localization of the endophyte was

recorded by two investigators: OSRORN (1909), found irregularly arranged bunches of lateral roots on Amyelon that resembled coralloid mycorrhizae; and on sectioning, found that there was a thick cortex which was divisible into two regions, the inner cortex which contained dark cells that showed evident fungal hyphae, some having terminal vesicles. These data were confirmed by HALKET (1930), who described a definite “fungal zone” about the stele. His description and excellent photomicrographs indicate that these Cordaites rootlets were similar in structure to living coniferous rootletp. The septate hyphae were mainly inter- cellular hut formed vesicles and arbuscles intracellularly ; while digestion cells wore found in cortical cells. The vascule was never invaded.

Coming next to living Gymnosperms, i t is remarkable that there is so little anatomical information about their mycorrhizae extant. I n fact, there is really not a single first-class anatomical study of any gymnospermous mycorrhim in print, and there are only a few incidental studies of them. Here is a field for some anatomist ! Of those who have recorded localization of the endophgte in Gymnosperms we may cite the following :

Two species of Taxus, T. baccata and T . canadensis, were reported by PRAT (1920, 1934) as localizing the endophyte. The former “normally develops” an endotrophic mycelium in the absorbing system of the roots, in the cortex only, and never penetrates the central cylinder, which “is protected by a tannin-filled endodermis”. Analagous structure, said PRAT, was found in roots of Torreya and Cephalotaxua. Tn the Canada yew, the apical meristem is never infected: infected regions are swollen and tuberous, and there are arhus- cles but rarely vesicles in infected cells, while pbgocytosis occurs. “Symbiosis is scarcely the term to be used, but disease; for the invading fungus is limited by the host.” REINKE (1873) had said that there is infection in the inner cortex of T . baccata and of Cephalotaxua; while in Torreya nucifera the endophyte occupied 2-3 lagers of outermost cortex.

I n an early study of coniferous rootlets, NICOLAI (1865) described a “peculiarity” of these rootlets in that the outermost cortical layer was transformed by “curious thickenings” of the cell-wall; or in other words, by HARTIQ net. A few years later, v. TIECHEM (1870) noted that in Pinus pinaea and other conifers, the penultimate layer of root cortex was filled with a solid matter. Various other gymnospermous mycorrhizae were reported on by REINKE (1873), who said that infection could be traced forward t o near the apex; also that hyphae are found in Biota orientalis - cells next to innermost layer of cortex, and similarly in Thuja occidentalis; Cupressus sempervirens, in Ginkgo biloba - 2-3 layers of outermost cortex, in Podocarpue neriifolia - all the cortex, in Sequoia gigantea - several layers of inner cortex, in Fremula rhomboidea - cortex but never central cylinder.

NOELLE’S (1910) studies of coniferous mycorrhizae were largely made from pot cultures; and he had these items to report: Only in the Abietineae are there typical ectotrophic mycorrhizae, whereas in the Araucarineae, Taxoideae, and Cupressineac, there are endotrophic mycorrhizae with the endophyte in the primary cortex. Of particular interest are those cases, as Cunninghamia, in which the hyphae penetrate only a few certain cell layers without any reason being apparent why they should not penetrate all the cortical cells. Entry of the endophyte causes the cortex t o enlarge greatly, and the stele to become monarch.

I n Pinus, MELIN (1921) found that his several strains of “Mywlium radicis” at first grew intracellularly in the outer cortical cells where they formed a pseudoparenchyma, later forming an HARTIC net and mantle. Pines on whose roots truffles were found were seen by REESS (1880) to possess hyphae that penetrated intercellularly in the root cortex. In the Monterey pine, MACDOUCAL (1943) discovered that fungi infest only the cortex.

Summarg Mycorrhizae of gymnosperms have been little studied by anatomists ; but the small amount

of data extant upon them indicate that here there is the same sort of localization of the endophyte as one finds in other plant groups. The fungus penetrates not further than the inner cortex, while the stele and the growing point oE the root are free from infestation. 12 2 eltschrift f. Allg. Mikrobiologie

170 ARTRUR PIERSON KELLEY

In various gymnosperms, there is localization of the endophyte in certain layers of the cortex. Even in the fossil Cordaites there is the same sort of localization of the endophyte in the cortex, and the stele is never invaded.

L i t e r a t ur e ci t a t i o n s H A L I ~ T ~ T , 1930. The rootlets of “Amyelon radicans”, WILL. ; their anatomy, apices and

MACDOUGAL, D. T., 1943. Study of symbiosis of Monterey pine with fungi. Yearbk. Amer.

MELIN, E., 1921. tfber die Mykorrhizenpilzen von Pinus sylvestris L. und Picea Abies (L.)

NICOLAI, 0. C. R., 1865. Das Wachstum der Wurzel. Schriften d. k. physikalisch-okonom.

NOELLE, A. 0. W., 1910. Studien zur vergleichenden Anatomie und Morphologie der Koni-

OBBORN, T. G. B., 1909. The lateral roots of Amyelon radicans WILL. and their mycorhiza.

PRAT, H., 1926. Etude des mycorhizEs du Taxus baccatn. Snn. Sci. nat. Rot. 1Ome. s8r.

-, 1934. Les mycorhixEs de I’if du Canada (Z’a‘axu~ canadensis MARSH.) Naturliste Canad.

REESS, Y., 1880. tfber den Parasitismus von Elaphomyces granulatus. Sitz. ber. d. phys.

REINKE, J . , 1873. Morphologische Abhandlungen. viii, 122 pp. Leipzig, Wilhelm Engle-

v. TIEGHEM, P., 1870/1871. Recherohes sur la symetrie de structure des plantes vasculaires.

endophytic fungus. Ann. of Bot. 44, 865-905.

Phil. SOC. 170-174.

KARST. Vorlaufige Mitteilung. Rvensk. Bot. Tidskr. 15, 192-203.

Gesell. z. Konigsberg. Abhand. 6, 33-78.

ferenwurzeln mit Riickeicht auf die Systematik. Rot. Zeit. 88, 169-266.

Ann. of Bot. 23, 603-611.

8, 141-163.

61, 47-56,

med. SOC. z. Erlangen. Heft 12. 103-107.

man.

Ann. d. Sci. nat. Rot. 5me. s h . 13, 1-314.

I V . Localization of the endophyte in Angiosperms I n order t o treat this large topic, the siibject matter will be divided into five parts,

namely: Woody plants, hraths, herbaceous angiosperms, cxcepting - grasses, and orchids.

L o c a l i z a t i o n in woody A n g i o s p e r m s The Casuarinas possess nodulous root-bunches which MIEHE (1918) said contained a

mycorrhizal fungus; and tha t this fungus heavily infested the cortex but was never found in the vascular bundle. This “small hyphal fungus’’ may have actually been one of t h r bacteria: a t least, MCLUCKIE (1923) calls them bacteria (of the Pseudomonas type) with bacteroids in a “basal zone”. The meristem contains no bacteria but these were confined to a rortical zone.

The mycodomatia of Alnus are only partially mycotrophic because they seem primarily bacterial; yet WOLPERT (1909) described nodules of A . alno-betula as mycorrhizal, finding such structures even on seedlings. The meristem is free from infection, while hyph81 penetration of the cortex is through 1-6 cell layers, the hyphal branches ending in bladdery swellings, while coils may fill the whole cell lumen. These structures appear to be digested.

Mycorrhizae of CupuliEers were made notable by the work of FRANK (1885), although there is not so much detailed information about their anatomy in his paper; yet he did say that the hyphae never quite grow into the innermost layers of cortex. This condition was reaffir- med by AIANcm (1898), who inRisted that the fungus never penetrated into the cells but was strictly ectotrophic, a conclusion attested by LESSMAN (1928), who said that the fungus was kept out by a deposit in the cell-walls.

I n Ficus, REISSEK (1847) found “masses” (evidently fungal products) in outer root cortical cells; also in Peperomia. The endotrophic Iungus lives almost exclusively in cortical tissues of Hevea rubber trees, said D’ANGREMOND and VAN HELL (1939) while JANSE (1897) Rtated that in Ficus the fungus penetrates directly to the second layer of cortex. I n various niem- bers of the Rosales ROULET (1910) found the habit of the endophyte remarkably uniform: the mycelium traverses the piliferous layer, penetrating into the cortical cells where it ramifies but seldom penetrates further than s/, the width of the cortex. The mycelium exhibits altprations of form and structure as i t approaches the endodermis, which is never penetrated. Degeneration of the hyphae is most abundant in the vicinity of the endodermis.


I n Eheocarpus (JANSE, 1897) the fungus develops only in the mediocortex: in Begonia robusta the fungus inhabits only the distal part of the root: in Caesaria and Pithecolohium, the fungus never enters tannin cells.

KLECKA and VUROLOV (1935) stated that disintegration in ectotrophic mycorrhizee occurs particularly in the neighborhood of the endodermis. I n endotrophic m ycorrhizae the disintegration is concentrated about the vascular bundle.

With Eucalyptus (SUITII and POPE), 1934), the fungus is usually present inside cells of the epidermal layer and the outermost cortical layer, but rarely occurs in 'any deeper layer.

I n Pitis (PETRI, 1907), the fungus penetratcs the epidermis in rear of the nodosity and spreads in the cortex.

I n Cacao (PYKE, 19351, infection is conifined to cortex of tip portion of the root, the ccn- tral cylinder and the long roots being free from infection.

Citrus is mycorrhizal, REED and FR~MONT (1934) finding the mycelium limited t o the cortex by digestion.

L i t e r a t u r e c i t a t i o n s BOULET, V., 1910. Sur les mycorhizbs endotrophes de quelquer arbres fruitiers. Comt.

rend. Acad. Sci., Paris. 160, 1190-1192. D'ANOREMOND, A., 1939. Mycorrhiza von Hevea brasilienais MULL.-ARU. Repr. from Versl.

Ver. Proefst. Personeel. Medan-Sumatra. 16 pp. W. F. VAN HELL, joint author. FRANK, A. B., 1885. Uber die auf Wurzelsymbiose beruhende Ernahrung gewisser BIume

durch unterirdische Pilze. Ber. d. deut. bot. Ges. 3. 128-145. JANSE, J. BE., 1897. Les endophytes rodicaux de quelques plantes Javanaise. Ann. d.

Jard. bot. d. Buitzenzorg. 14, 53-201. KLECHA, A., 1935. Sronavaci studie o mykorrhize dievin (uiitkovyah. okrasnych a ovo-

cfnch). Sbornik bskoslov. Akad. ZemBd. 10,443-457. V. Vukolov, joint author. LESSMAN, L. L., 1928. A new form of ectotrophic mycorhiza. Trans. Illionois Acad. of Sci.

MCLUCKIE, J., 1923. Studies in symbiosis IV. The root nodules of Casuarina cunninghamia and their physiological significance. Proc. Linnaean SOC. of New South Wales., n. s . 48,

MIEHE, H., 1918. Anatomische Untersuchung der Pilzsymbiose bei Casuarina epuisetifolia nebst einigen Bemerkungen uber das Mykorrhizenproblem (Festschrift zum siebzigsten Geburstage von ERNST STAHL) Flora 111/112,431-439.

PETRI, L., 1907. Sulle micorize endotrofiche della .vite. Atti. Reale Accad. dei Lincei. Cle se di Sci. Fis., Math. e Nat. 5a ser. 16 (l), 789-791.

PYRE, E. E., 1935. Mycorrhiza in Cacao. Ann. Rept. Cacao Res., Trinidad 4 (1934), 41 - 48. REED, H. S., and T. FRBMONT, 1935. Factors that influence the formation and development

of mycorrhixal associations in Citrus roots. Phytopath. 25, 645-647. REISSEK, S., 1847. IV. Uber Endophyten der Pflanzenzelle, eine gesetzmafiige den Samen-

faden oder beweglichen Spiralfasern analoge Erscheinung. Naturw. Abhandl., herausge- geben von WILHELM HAIDINOER. 1, 36-46.

SMITH, N. J. G., and F. B. POPE, 1934. The association between the Gasteromycete Poly- sacchum and Eucalyptus roots. Trans. Brit. Mycol. SOC. 19, 95.

WOLPERT, J., 1909. Vergleichende Anatornie und Entwicklungsgeschichte von Alnua alno-betula und Betula. Flora 100, 37-67.

20, 80-81.

194- 205.

L o c a l i z a t i o n i n h e a t h s Monotropa fascinated investigators for years because of its supposed parasitism; and it

remained for KAMIENSKI (1884) to elucidate its true nature. He said that the fungus of Monotropa lives on the surface of the root and does not penetrate the cells except that sometimes in older portions it invades those which are filled with a brown content (tannin). The fungus always develops where tissues are maturing and diminishes toward the apex. PEKLO (1908) disagreed about tannin, saying that great tannin vacuoles set bounds to the fungus; but FRANCKE (1934) said that tannin content of epidermal cetls is no protection against ingress of the fu gus; he also found hyphae in epidermal but never in deeper layers.

I n PiroZa, mycelium is spread over the whole length of the root ( ~ T H 1920), but is confined to the epidermal cells, in which digestion stages appear, Several species of Pirola were found to be rich in phlorsglucotannins. P. rotundifolia forms tubers (KRAMAR, 1899) There is no fungal invasion of the root-tip, and there is never penetration of the sub-epi- 12.


dermal cells. “These mycorrhizae”, he said, “are interesting because they combine features of the ectotrophic and endotrophic sorts.’’ (And ectendotrophy was discovered later!)

With Arbutus, RIVETT (1924) noticed that peripheral cells, except a t the growing point are to be found filled with partially digested hyphae. I n many branches formed late in the season, hyphae penetrate more deeply into cortical cells which take on the same appear- ances as those on the periphery. More rarely the growing point ceases functioning and the whole tissue of a branch becomes infected without any formation of vascular tissue. In 1917, DUFRENOY had also stated that the fungus invades external layers of cortex; and he found an unstable relation between fungus and host: thus, he said that where invasion is checked a tubercle is developed, whereas in other cases the fungus spreads through all the tissues of root, stem, leaf, and fruit; and after death of the plant, the fungus continues to live as a saprophyte.

Calluna (of RAYNER 1913) has branched hyphae projecting from the surface of the mycorrhiza; and many of the cortical cells contain characteristic “knots” of mycelium. Later, 1915, she said that the fungus grows throughout the whole plant, but does not grow into the embryo.

In Rhododendron javanicum, the fungus develops only in the epidermis (JANSE, 1897); and it is thus also in Vaccinium. But RAYNER (1929) stated that she could not secure a fungus-free of the plant latter genus, because the fungus penetrated all tissues of the host, even the ovary. ADDOMS and MOUNCE (1931) said that hyphae penetrate epidermis and cortical parenchyma; while in the stem, infection is greatest in pith and cortex (or, in other words, the tissues that are the least active).

L i t e r a t u r e c i t a t i o n s ADDOMS, R. M., 1931. Notes on the nutrient requirements and the histology of the cran-

berry ( Vaccinium macrocarpon AIT.) with special reference to mycorhiza. Plant Physiol. 6, 653-668. I?. C. Mounce, joint author.

~ ~ F R E N O Y , J., 1917. The endotrophic mycorhiza of E r i c a c ~ a ~ . New Phytol. 16, 222-228. PRANCKE, H. L., 1934. Beitrage zur Kenntnis der Mykorrhiza von Monotropa Hypopitys L.

FI~RTR, P., 1920. Zur Biologie und Mikrochemie einiger Pirola-arten. Sitzungsber. Math.

HASSELBAUM, G., 1931. Cytologische und physiologische Studien zur ericoiden endo-

JANSE, J. M., cf. p. 168. KAMIENSKI, F., 1884. Les organes regetatifs du Monotropa Hypopitys. Mem. d. 1. SOC.

KRAMAB, U., 1899. Studie o mykolize u hrugtiEky okrouhloiste (Pirola rotundifolia L.).

PEKLO, J., 1908. Die epiphytischen Mykorrhizen nach neuen Untersuchungen. I. Hono-

RAYNER, M. C., 1913. The ecology of Calluna vulgaris. New Phytol. 152, 59-77. -, 1915. Obligate symbiosis in Calluna vulgaris. Ann. of Bot. 29, 97-133. - , 1929. The biology of fungus infection in the genus Vaccinium. Ann. of Bot. 43, 55-70. RIVETT, M. F., 1924. The root tubercles of Arbutus Unedo. Ann. of Bot. 38, 661-677.

Analyse und Synthese der Symbiose. Flora 129 (n. s., 29). 1-52.

Naturw. K1. Akad. Wiss. Wien. Abt. 1. 129, 559-587.

trophen Mycorhiza von Empetrum nigrum. Bot. Arch. 31, 386-440.

Nat. e t Math. de Cherbourg. 24,5-40. (Title page bears date of 1882.)

Bull. inter. d. 1’Acad. d. sci. d. BohBme. 8, 9-15.

tropa Hypopitys L. Bull. inter. de 1’Empereur Fr. Josef. 13, 87-107.

L o c a l i z a t i o n in c e r t a i n h e r b a c e o u s A n g i o s p e r m s -1ccording to ScHLIcriT (1889), infections of Ranunculus ncris is very constant in side

roots but not in the main roots, the fungus penetrating the epidermis intercellularly to the innermost cortex where most of the hyphae are found within cells, the inner hyphae being continuous with a mantle about the outside. The same sort of structure was found in other Ranunculi.

I n roots of Sempervivum (being two species from the Vienna Botanic Garden), normal cells near the root-tip were seen to be without fungal infection. Infected cells became enlar- ged up to 10 times the normal size. (ZACH 1909)

Strawberry roots in Scotland, said O’Brien (1928), possess a inycorrhizal fungus which lives in localized patches of the inner cortex (which are figured one cell layer removed from the endodermis). Arbuscles are formed intracellularly and starch disappears.

Cotton possesses mycorrhizal fungi (SABET, 1939) which penetrate two or three layers of cortical cells, then enter the cells and form the “arbuscle-sporangiole apparatus”, especially within the cortical cells next to the endodermis.

Localization of the endophytc 173

A study of smaller roots of celery, according to Rawlins and SMITH (1925), showed that they were heavily infested with a mycorhizal fungus which was confined t o cells of the inner cortex.

Nodules are found on roots of Datisca, said TROTTER (1900), and within them is a fungus that is contained in a definite zone of cortical cells.

The fungus of Gentiana Clusii enters the cortex where almost every cell is invaded and hyphal coils are formed (SCHIMNLER, 1937). A1 0, in Obolaria, aid PERROT (1898), “in the cells of the cortex which persist, one finds the hyphae of the fungi”.

In Lobelia (FR.4SER, 1931), the apex is always free from infection but back of the apex there are three zones: of fungal invasion, of fungal enlargement (middle cortex), and of fungal depletion.

Leontodon shows typical structure of a n endotrophic mycorrhiza in finer parts of the root system, the cortical cells being filled a n homogeneous substance, even the newly formed cells beside the endodermis (SCHLICHT, 1889).

I n the Burmanniaceue there is also localization of infection. The rare Sarcosiphon of Australia contained fungal hyphae between cortical cells and around the stele (COLEMAN, 1936). A line drawing shows hyphae coiling in the first exocortical layer; while hyphal knots and digestion stages are suggested in immediately subjacent cells. JOHOW (1889) said that the central cylinder is free from infection, which is chiefly limited to individual peripheral cells, in the BeLrmanniuceae that the studied. I n Thiamia (PFEIFFER, 19141, the tip region is free from invasion, most of the cortex being infested but a few cells outside the endodermis are free.

In Philesia, a liliaceous plant, MACFARLANE (1897) found a fungus forming an abundant growth in the mesocort x to the 10th t o 12 t h layer of cells back of the apex. Invariably crystal cells were left untouched.

I n Iris, the fungus was found in cortcx only, fruiting bodies being figured for cortical cells. (NAEQELI, 1842).

L i t e r a t u r e c i t a t i o n s COLEMAN, I). G., 1936. Sarcosiphon Rodwuyi in Australia. Victorian Xat. 52, 163-IBG. FRASER, L., 1931. An investigation of Lobelia gibbosa and Lobelia dentata. I. Mycorrhiza,

latex system and general biology. Proc. Linnean SOC. New South Wales. 66, 497-525. JOHOW, F., 1885. Die chlorophyllfreien Humusbewohner West-Indiens, biologisch-morpho-

logisch dargestellt. Jahrb. f. wiss. Bot. 16.415-449. MACFARLANE, J . M.. 1897. A mycorhiza in the roots of the liliaceous genus Philesia. Bot.

Gaz. 26, 106-107. NAEQELI, K. W., 1842. Botanische Beitrage. 7. Pilze im Innern von Zellen. Linnaea 16.

O’BRIEN, D. G., 1928. Disease in strawberries. Scottish Journ. of Agr. 11, 286-297. PERROT, M. E., 1898. Anatomie comparke des Gentianckes. Ann. d. Sci. nat., Bot. VITT.

PFEIFFER, N. E., 1914. Morphology of Thismia americana. Bot. Gaz. 67, 122- 135. RAWLINS, T. E., 1925. A mycorrhizal fungus found in the smaller roots of celery. Phyto-

path. 16, 727. E. H. SMITH, joint suthor. SABET, Y., 1939. Cotton mycorrhiza. Nature. 144, 37. SCHIMMLER, G., 1937. Uber Pilzwurxeln an Enzia-gewiichsen. Blumen- u. Pflaneenbau

v. m. Gartenwelt. 41, 161 - 162. SCHLICHT, A. E. C., 1888. Uber neue Falle von Symbiose der Pflanzenwurzeln mit Pilzen.

Ber. d. deut. bot. Ges., 6, 269-272. TROTTER, A., 1901. I micromiceti delle Galle. Atti. Reale Inst. Venetio. 1900. cf. Rev.

ZACH, F., 1909. Untersuchungen uber die Kurzwurzeln von Semperaivum und die daselbfit auftretenden endotrophe Mykorrhiza. Sitz. ber. d. k. Akad. d. Wiss. z. Wien. Math. Naturw. K1. 318 (Abt. l), 185-200.

278 - 285.

7, 105-292.

Mycol. 23, 30-31.

L o c a l i z a t i o n i n c e r t a i n Cramineac A microscopical examination of roots of sugar-cane (Saccharum) made evident the pre-

sence of two parasites, one a Nematode, the other a fungus. The fungus bores through outer cell-walls of the root and fills many of the cells within, but never penetrates the central cylinder. (TREUB, 1885).


Holcus lanatw, in young roots, always has endotrophic mycorrhizae which are continuous with the external fungal layer. I n the cortex, the hyphae form a mantle about the endodermis which often extends almost to the root-tip. Fungal masses are found only in the sub-epidermis (SCHLICHT, 1889).

Emmer (Triticum dicoccum) caryopses from an Egyptian tomb, age about 4000 years, showed structure like grain of today: Fungal hyphae were found between the seed-coats and the aleurone layer, without any extension, either outward or inward. All seeds were infected. (LINDAU, 1904).

It is Lolium that has attracted the most attention, among the grasses, to fungal infection of the grain. LINDAU (1904) found exactly similar structure in both Triticum and Lolium. VOQL, in 1897, discovered an fungus in the hyaline layer of Lolium seeds (said HANAUSER, 1898); and attributed the poisonous properties of the grain the the fungus. HANAUSEK found infection in all sound grains of L . tementulum but not of L. perenne: the fungus formed a layer within the seed coat, next the aleurone layer. A more detailed study was made by NESTLER (1898), who considered that localization of the fungus in the caryopses is due t o pressure of the developing embryo against the nucellus, which contains the fungus. L. tementulum grains were further studied by FREEMAN (1903). who found hyphae in a broad “infection layer”, directly in contact with the embryo, which is penetrated by hyphae from this layer. Under favorable conditions, hyphae may penetrate the embryo to within 1-2 cells of the tip. Still further studies were made by MCLENNAN (1920), who found the endophyte in the aleurone layer and penetrating the scutellum. FREEMAN’S infection patch was not found, but rather penetration of the scutellum from the aleurone wherever the two were in contact.

MCLENNAN (1926) also reported on mycorrhizae in Lolium : I n cortex, the fungus spreads in the cells but not air chambers, and i t is also localized horizontally. The cortex is divisible into three regions: Outer layer, 2-3 layers with spread hyphae, inner seat of interchange. There is a considerable discussion of the interchange, but no mention of any penetration of the ventral cylinder by hyphae.

L i t e r a t u r e c i t a t i o n s FREEMAN, E. &I., 1903. The seed fungus of Lolium tementulum L., the darnel. Phil. Trans.

of the Roy. SOC. of London. Ser. B. 196, 1-27. HANAUSEK, T. F., 1898. Vorlaufige Mitteilung uber den von A. VOQL in der Frucht von

Lolium tementulum entdeckten Pilz. Ber. Dtsch. bot. Ges. 16,2-3-207. LINDAU, G., 1904. Uber das Vorkommen des Pilzes des Taumellochs in altiigyptischen

Samen. Sitzber. d. kgl. PreuO. Akad. d. Wiss. 1904 (2), 1031-1036. MCLENNAN, E., 1920. The endophytic fungus of Lolium. Proc. of the Royal SOC. of Victoria.

n. s., 32(2), 252-301. NESTLER, A., 1898. uber einen in der Frucht von Lolium temulentum vorkommenden Pilz.

Ber. Dtsch. bot. Ges. 16, 207-214. SCHLICHT, A. E. C., 1889. Arbeiten a m dem pflanzenphysiologischen Institute der Konig-

lichen landwirtschaftlichen Hochschule in Berlin. XIII. Beitrage zur Kenntnis der Ver- breitung und der Bedeutung der Mykorrhizen. Landwirt. Jahrb. 18,478-506.

TREUB, M., 1885. Onderzockingen over serehziek Zuikerriet in’s lands plantentium te Buitenzorg. Meded uit’s lands Plantentium, no. 2. 59 pp. Batavia, Landsdruckkerij.

L o c a l i z a t i o n i n s o m e o r c h i d s Neottin was the orchid which first received attention from mycotrophists, probably

because it is a non-green saprophyte. PRILLIEUX (1848) found the root cortex fungal- invaded and separable into three regions, the innermost layer having cell-content mixed with distinct “fibers”, the intermediate lagers comprised almost entirely of interlaced “fibers”; but REISSEK (1847) had already shown the fungal character of these “fibers”, not only in Neottia but in other orchids. He said that in Neottia hyphae stray into the innermost cortical cells; in Orchis Morio, it occurs in all or in most of the cortical cells; in Goodyera the cells are filled with a yellow granular mass next to the vascular cylinder. The presence of an abundant coloured material in determinate cell layers of the cortex was commented on by DRUDE (1873), who found that hyphae first appear in the outer two layers of cortex a t a little distance from the root apex, and t h a t they always maintain this distance in later growth of the root. This constant entrance of a parasite into definite layers of the subter- ranean organs requires closer consideration: It was never found in the epidermis, while


presence of mycelium in cortical layers seems to signify that here there is greatest concentration of organic soil substances. The most detailed study of Neottia was made by MAUNTJS (1900): He said that the 3-4 outermost layers of cells beneath the epidermis are completely and without exception inhabited by the fungus while in rhizome and stem, 6 layers even may be infected. The fungal infected cells were distinguished as Pilzwirtszellen u. Ver- dauungszellen, terms which MAUNIJS devised : The digestion-cells take the outer and inner layers while the fungal-host cells occupy the middle layer of Nwttka-cortex. No mention is made of stellar invasion, the fungus being confined to the cortex.

While Neottia was being investigated, EIDAM (1879) commented on the constant occurrence of fungal infection inGerman orchids, the hyphae filling cortical cells all the way to the central cylinder. MOLLBERU (1884) investigated a couple dozen species, both native German orchids and exotics, and found that hyphae usually penetrate into the middle fundamental tissues; also, that some roots are free from infection. WAHRLICH (1886) claimed to have investigated 500 exotic orchid species, and stated that only the epidermis, exodermis and middle cell layers of cortical parenchyma are infected, while the other tiesues are fungus-free. Also, the raphide-containing “slime-cells” and the 2-3 layers next to the vascular bundle are fungus-free. The relationships in the exotic orchids seemed to be the same as in native ones. Another study of tropical orchids was made by DIETZ (1930) a t Buitzenzorg; he found that fungal infection of the orchid roots was general although the mode of infection, and its degree and localization varied for most species, the mycelium being present in the whole or a sectorial portion of the cortex, but only in fleshy roots and not entering very deeply. Also a t Buitenzorg was JANSE (1897) who found that the endophyte always entered the cortex through “cellules de passage”, proceeding to the 2d layer of the cortex while in the 3d layer vesicles form on hyphal tips. Within this layer the cortical cells contain much starch.

BUROEPF (1909) also testified to localization of the endophyte: The mycotrophic fungus enters through root-hairs, he said, into the most external cells, and penetrates to the endodermis, dissolving whatever starch is present as it goes; then hyphae ooil together in root cells and protoplasm of the cells begins to digest it.

American orchids were reported by HOLM (1904) : In Cypripedium, hyphae penetrate no further than cortex in one species, into endodermis and pericambium in 2 species. In most species hyphae were present in cortex, and in no oase was infection reported for xylem or phloem. He reported a facultative infection for Spiranthes, confirmed by AMES (1921), who found the fungus filling cells of cortex about the vascular cylinder. A longitudinal section of the root shows that certain areas of the root have a capacity to repel advance of the fungus: “It is as if there were some fungicidal capacity in cells of the root structure that restricts the fungus to a limited area.”

In Ophyre (DANUEARD, 1900), the cortex but never the central cylinder is infected, great coils being formed in cells removed 2-3 layers from the surface. The fungus never penetrates raphide cells.

In Pogonia, CARLSON (1938) reported that the fungus enters the epidermis, passes through 1-2 layers of cortical cells, then forms a tangled mass of hyphae in the next several layers. Starch disappears in the cmtex after the fungus enters. The fungus seems never to penetrate deeper than the inner cortical cells.

In Galeola of Japan, the symbiont ipvades the cortex during summer and autumn, while ingestion proceeds through the winter. As with many other orchids, the process of ingestion of the fungal coils is preceded by a turning point a t which the h. i. c. of the hyphal clumps reaches a maximum of pH 6.2 (HAMADA, 1939).

In Cornlliorhiza JENNINQS and HANNA (1898) saw that the fungus went through outermost layer of cells to a zone in which the hyphae coil within thin-walled cells. There is a paucity of starch in this zone; but within is a third zone in which starch increases in quan- tity as hyphae become less numerous. This orchid, said MACDOIJOAL (1899), is infested early with a fungus that fills the mediocortex, and grows forward with the apex. THOMAS (1893) had earlier stated that hyphae in this orchid nre confined to a certain region of the stem, and are seldom found within 3 -4 mm. of the tip. The calls nearest the tip, in which they appear, contain but R few while farther back the hyphae increase in abundance. They penetrate cell-walls and fill every cell in a narrow 70ne of plerome. Still earlier, REINEE (1873) had found a fungus in the outermost cortical cells of this orchid, while the “slime” (digestion) cells form a cylinder, although one broken a t places and of varying width. MARUIJSE (1902) a180 spoke of the endophyte forming a layer of fungus-containing cells about the central strand.

176 ~\RTIIUR PIERSON KELLEY

In Gastrodia, the fungus penetrates into the cortex while branches of the hyphae are sent to a small localized region. both radially and tangentially; and the fungus is limited to a certain area about the infected spot. Three regions may be noted in the cortex. the third and innermost being where hyphae are digested by the host. Starch grains disappear before the invading fungus (KUSANO, 1911).

I n Cul!ypso ( M A C ~ O U G A L , 1899), the fungus lives in the outer cortex while inner cortex and apex are free from infection.

In Narcodes (OLIVER, 1890), the fungus is confined to the surface of the orchid root and to the epidermis, never entering the epidermal cells nor penetrating lower than the epidermis.

I n Epitrhizanfhes, PENZIQ (1901) found yellow refractive bodies which he considered of fungal origin in the inner cortex also, in parenchyma cells of fundamental tissue and in scattered cells of endodermis.

In Rhizanthrlla (PITTIAN. 1929), it was found that with the exception of the epidermis, the outermost cells to the depth of about ten cell-layers contained an endotrophic mycorrhiza.

I n Dipodiiunz, a non-green orchid of Australia, MCLVCKIE (1922) discovered that the root cortex contains an endophpte, which is digested by the host. The fungua does not enter raphide-cells nor the mrristematic zone of the root.

Summar?! Angiospcrrns exhibit the same sort of localization of the endophyte in mycotrophic

structures that we have seen in other plant groups. In all cases noted, the fungus is confined to certain areas of root or rhizome tissue, never progressing further than a dcfined layer of cells. Tn no case were raphide-containing cells invaded. I n no case was meristernatic tisme of the apex invaded. In no case were the conduction tissues of the central cylinder invaded.

The matter is very clear-cut. I n all instances the mycotrophic fungus is checked in its growth through the host tissues: this checking is always at a definite, given, place in the plant’s anatomy; and the limitation serms due to the vascular plant.

L i t e r a t u r e c i t a t i o n s Anms, O., 1921. Notes on New England orchids. I. Spiranthes. Rhodora. 93, 73-85. BURQEFF, H., 1909. Die Wurzelpilze der Orchideen, ihre Kultur und ihr Leben in der

Pflanze. iv, 220 pp. Jena, G. Fischer. CARLSON, M. C., 1938. Origin and development of shoots from the tips of roots of Pogonia

ophioglossoides. Bot. Gaz. 100, 215- 225. DANQEARD, P. A. C., 1898. Observations de biologie cellulaire. Mycorrhizes d’0phrys

arachniferu. Rev. Mycol. 20, 13-18. DIETZ, J., 1930. Morphologisch-anatomische Untersuchungen der unterirdischen Organe

tropischer Erdorchideen Ann. d. Jard. Bot. d. Buitenzorg. 41, 1-26. DRUDE, O., 1873. Die Biologie von Nonotropa Hypopitys L. und Neottia nidzw-avis L. unter

vergleichendem Hinzuziehen anderer Orchideen. 68 pp. Gottingen, W. Fr. Kaestner. EIDAM, E., 1879. uber Pilzentwicklung in der Wurzel der Orchideen. Jahresber. d. bot.

Section d. Schleswigs. Gesell. f . vaterlandische Kultur. 67, 297. HAMADA, M., 1939. Studies iiber die Mykorrhiza von Galeola septentrionalis REICHB. Ein

neuer Fall des Mykorrhizabildung durch intraradicale Rhizomorpha. Jap. Journ. Bot.

HOLM, TH.. 1904. The root structure of North American terrestrial Orchidcne Amer. Journ.

JANSE, J. M. cf. p. 168. JENNIKGS, A. V., 1898. Coralliorhiza innata R. BR., and its mycorhim. Sci. Proc. of the

KUSANO, S., 1911. Gastrodia eluta and iLs symbiotic association with Armzllaria mellea.

MACDOUQAL, D. T., 1899. Symbiotic saprophytism. Ann. of Bot. 13, 1-47. MCLUCKIE, J., 1922. Studies in symbiosis. I. The mycorrhixa of Dipodiuwz punctatum K.

MAGNUS, W.. 1900. Studicn an der endotrophen Mykorrhiza von Neotlia nidusavis L.

MARCUSE, M., 1902. Anatomisch-biologischen Beitrag zur Mykorrhozenfrage. Thesis

10 (1/2), 151-211.

Sci. 4th ser. 18, 197-211.

Roy. Dublin SOP. 9, 1-11.

Journ. Coll. of Agric., Imp. Univ. of Tokyo. 4, 1 - 66.

BR. Proc. Linnaean SOC. of New South Wales. n. s., 47, 293-310.

Jahrb. wiss. Bot. 35, 205-272.

Jena 35 pp.

Localization of the endophytc 177

MOLLBERO, A., 1884. Untersuchung uber die Pilze in den Wurzeln der Orchideen. Jenaiache

OLIVER, F. W., 1890. On Sarwdea sanguinea, TORR. Ann. of Bot. 4,303-326. PENZIG, 0. A. J., 1901. Beitriige zur Kenntnis der Gattunq Epirrhozanthes BL. Ann. Jard.

bot. Buitenzorg. 17, 142-170. PITTMAN, H. A., 1929. Note on the morphology and endotrophic mycorrhiza of Rlrizan-

thella gardneri, ROGERS, and certain other Western Australian orchids. Journ. Roy. SOC. West. Australia 15, 71 -76.

, PRILLIEUX, E. N., 1856. De la structure anatomique et du mode de vegetation de Neottia Nidua-avia. Ann. d. Sci. nat., Bot. 4me. s8r. 6, 267-282.

REINKE, J., 1873. Zur Ksnntiis dar Rhizome V O I Coralliorhdza u-d Epipogen. Nora. n. s.,31, 145-152; 161-167;177-184;209-224.

REISSEK, S. cf. p. 171. THOMAS, M. B., 1899. Some problems in Coralliorhiza. Proc. of the Indiana Acad. of Sci.

WAHRLICH, W., 1886. Beitriige zur Kenntnis der Orchideenwurzelpilze. Bot. Zeitg. 44,

Zeits. f. Naturwiss. 17, 519-536.

145.

480-487, 497-505. See also, Rev. Mycol. BO, 1- 10. 1896,

V . The cause of endophytic localization We may now inquire whether any reasons may be advanced for the curious Iocalization

which has been universally reported by investigators of mycotrophic organs for more than a oentury past. Our inquiries may be answered in part from the literature; and some of the data already presented may be aggregated in tabular form. Let us start with the apex of the root, and list what investigators have said about its relation to the endophyte: BARROWS (1935), Lycopodium: BRUCHMANN (1904), Ophioglosaum: CAVERS (1903), Mortoclea: E ~ A S E ~ (1931), Lobelia: FREEMAN (1903), Lolium: KAMIENSKI (1884), Nonotropa : KRAMAR (1899), Pirola: MACFARLANE (1897), Phileaia: PETRI (1907), Vktia: PFEIFFER (1914), Thismia: POULSEN (1886), Sciaphila: PRAT (1934), Pazus : REESS (1887), (General): REINKE (1873), Pellia: RIVETT (1924), AVhutU8: SCHLICHT (1889), Paris:

SPESSARD (1922), Lycopodium: SPRATT (1912), Elaeagnus: THOMAS (1893), Corallorhizu: WEST (1917), dlarattiacease: ZACH (1909), Craaaulaceae:

Here is decidedly a mass of evidence, from independent and noncollaborative sources, to show that the root-tip of vascular plants, and the growing point of thalli. is free from fungal invasion. And what portion of the root-tip? The growing point of the apex, the meristem, remains uninvaded. Consider the meristem more closely, for there are others who have noted that not only apical meriaterns but meristems in general are fungus-free: BERNATSKY (1900), Fungus penetrates suoh tissues as are not physiologically active. CHALAUD (1932), Progress of hyphal growth in hepatias is checked by active meristemrt-

tic cells. MCLUCKIE (1922), Fungus does not enter meristematic zone of root. MAQROU (1925), Fungus imhibited about developing archegonia and antheridia of

hepa tics. PRAT (1934), Fungus is limited by the groat phagocytotic power of the meristern. PYKE (1935) : Meristem of Cacao was never invaded WOLPERT (1909) : Meristem of Alnus root is free from infection

Fungus absent from apical regions Root-tip remains free Growing point not infected Apex of root always fungus-free Fungus grows to within 1-2 cells of tip Fungus diminishes towards apex No fungal infection of root-tip Hyphae 10-12 layers cells back of apex Fungus never extends to root-tip Tip region of rdot fungus-free Apex of root contains no fungus Tip of root iq fungus-free Healthy apex never invaded Vegetative point free No digestion stages a t growing point Infection a t Borne distance from tip Infection extends almost to tip Fungus 2 - 3 cell layers back of tip Bacteria found in region back of tip Seldom found within 3-4 mm. of tip Endophyte absent from tip Cells near tip without infection

Holcue :

178 A R T ~ ~ U R PIERSON KELLEY

It is evident that not only meristems, but green tissues, chlorenchyma, are also uninvaded by the endophyte; and this fact has been shown independently by several students: AURET (1930), Lunularia: Fungus is confined to definite zone below the assimilating

tissue. SPESSARD (1922), Lycopodiuin: Fungus coiled in lower part of prothallus; does not enter

palisade. STAHL (1942), Lunularia: Green tissues shunned by fungus ; In hepatics generally

it, is under portion of thallus that is infected. BURGES, (1939), Orchida: Root-cells containing chloroplasts uninfected: those

without them are infected : indicates formation of chloroplasts accompanied by immunity

GALLAUD is said to have remarked also that chlorenchyma repels fungus. Secretory cells, particularly raphide-cells, are uninvaded by the endophyte, as witness

the following: DANGEARD (1898), Ophyra: GALLAUD (1905), (general) : MACFARLANE (1897), Philesia: MCLUCKIE (1922), Dipodium: PFEIBFER (1914), Thismia: WAHRLICH (1886), Orchida:

Fungus never enters raphide-cells Secretion cells act to repel fungus Invariably crystal-cells were untouched Fungus does not enter raphide-cells Raphide-cells are free from fungus Raphide-containing cells are completely free

In the case of t ann in cells the evidence is similar, although a couple of investigators indicate invasion of tannin-filled cells by the fungus, but in this case the fungus is apparently a saprophyte : WEST (1917), Marattiaceae: Mycelium is always absent from tannin-cells, even when hyphae

are abundant in adjacent cells. ,,COOK [MEL. T. CWKJ has shown that tannin exerts a deleterious effect upon certain fungi; while GALLAUD remarks that fungi are in general repelled by secretory cells or by chlorenchyma".

KAMIENSKI, (1882), Monotropa : Fungus sometimes invades cells containing a brown content (tannin).

MANGIN (1910) : In later stages of tree mycorrhizae, as they break down, saprophytic fungi invade and digest the gum-tannin.

PEKLO (1908), Monotropa: Great tannin vacuoles set bounds to fungus. But in his 1909 paper on Betulaceae and Fagaceae PEELO said: The host-plant increases its tannin content as a protection against the invader; but the fungus becomes adapted to the condition and uses the tannin as food.

PRAT (1926), Taxus: A tannin-filled endodermis protects the central cylinder from invasion. This statement is repeated in PRAT'S 1934 paper.

REXHAUSEN (1920) : In pines, oak, Nonotropa, there is marked deposit of tannin in endodermis and epidermis as a protective device against the fungus. With regard to the cent ra l cylinder, we have seen a uniform and very general testi-

mony that the endophyte is checked in its growth a t a definite, set, distance from the stele. Some of the investigators that have especially stated that the stele is never invaded by a mycotrophic fungus are : BRUCRMANN, DANQEARD, HALKET, JOHOW, KAMIENSKI, MIEHE, PETRI, PRAT, PYKE, STAHL, TREUB, WAHRLICH.

Now why should the active, prosenchyma tissued be immune to invasion of the endophyte while inactive parenchyma is invaded 1

The first answer that suggested itself was, that the vascular plant produces a fungicide. This was the conclusion of the brilliant N O ~ L BERNARD, whose early death was such a loss to mycotrophic science. His views are explained in two papers presented in 1909, the former having been summarized by VUILLEMIN: In comparing the observations of JANSE, MOLLIARD, W. MAGNUS, GALLAUD, etc., in regard to the reactions of plant cells in presence of fungal or animal parasites, BARNARD sees the Rhizoctoneae and their orchids as two antagonists developing their means of attack and defence. Symbiosis represents an immunity attained by phagocytosis, the vascular plant making use of all its means of defense in order to pre- serve its essential tissues. The formation of mycelial coils in orchids, of GALLAUD'S arbuscles im nycorrhizae of Allium, of JANSE'S sporangioles in roots of Java plants, is considered as a phenomenon of agglutination due to a humoral property of phagocytotic origin. . . . The latter paper (a posthumous production from BERNARD'S notes) summarizes some experiments on orchid tubers: The experiments indicate (said the writer) the existence in the tuber of a substance capable of rapidly killing the protoplasm of the fungus, a rapidly diffusible


substance and hence probably a crystalloid; a substanoe comparable to a ‘diastase’, and destroyed by a temperature above 5 5 O .

BERNARD’S ideas were taken up by MAQROU, who related them to the theory of tuberisation. He said (1921): I n the case of symbiosis, toxic constituents of the cell-sap of the host- plant react in such a way that the fungus remains limited to certain parts of the plant where it distributes itself, peloton-like, in each cell. These coils are to be compared with the agglutination of bacteria in infected animals. Plants become immune to the fungus as shown by the fact that in host-plants of lessened vitality the fungus does not form coils but penetrates throughout the tissues. BERNARD showed toxic action of the cell-sap experimentally. .. The whole problem of symbiosis is accordingly reduceable to the question of physical- chemical modification of the cell-sap, which has the power to stop growth in thickness o r to accelerate i t ; and of the ability of the fungus to bring about this modifi ation.

AMES (1921) was also impressed withB~RNmD’s ideas, and said: In study (of orchid tubers) it is best tomakea longitudinal section whichthenshows that certain areas of the root have the capacity to repel advance of the fungus, and that in this respect the roots of #pi-- ranthee are comparable to bulbous thickenings of certain Ophrydeae studied by BERNARD, for “it is as if there were some fungicidal capacity in the cells of the root structure that restricts the fungus to a limited area.” Or, in Spiranthes we may have a plant which is able to defend itself against an intrusive fungus by means of a digestive process that protects tissues of vital importance. “In other words, there are two types of cells in the root system, - one type characterized by a capacity to digest the fungus and to hold i t in check; the other type found in infected regions and characterized by a capacity to act symbiotically with the invading fungus.”

Two sorts of resistance were also posited by BURGES (1939), a mechanical resistant-: by which the cell walls become thickened and yellow, and a protoplasmatic resistance in which the invading hyphae are completely broken down by what appears to be the action of proteolytic -my nes. When extracts of orchid tissues from tubers, stems, leaves and roots were added to young cultures of the endophytic fungus, there was inhibition of fungal growth and complet,e decomposition of hyphae within 3-4 days, root extracts being the least toxic. Sap of host cells, withdrawn by means of micro-pipette and added to blocks of agar smear cultures (of the endophyte) produced visible changes in the hyphae within 24 bours; and at the end of four days som empty hyphae could be seen. I n 1936, BURQES had said that the fungi concerned (in mycorrhizae) are weak pathogens whose activity is curbed by the reactions of the host cells. NOB~COURT (1923) was yet anbther disciple of BERNARD. Noting BERNARD’S demonstration of a fungicidal Substance in tubers of the Ophrydeae (Loroglossum hircin,um), the author repeated the experiment with the same orchid and with the fungus Orcheomyces psychodie BURQEFF and 0. ChlorantheBURaEFF. The orchid secreted a mucilaginous substance which killed the fungus in a zone of 5 mm. about the cut tuber. BERNARD found that boiled tuber no longer offered resistance to the fungus, and NOBECOURT found the same; and also that tubers exposed to a temperature of - 150 “C and to fumes of chloroform no longer offered resistance. It is unreasonable to suppose that a chemical substance should be more destructive by cold as well as by chemical action: There is here more the resemblance of a life phenomenon. “In our opinion, is i t not because the fungicidal substance is destroyed that the fungus is not arrested but because it (i. e., the fungicidal substance) has not been produced a t the moment of attack by the fungus, due to death of cells of the tuber. This substance, arising in the tubercle under influence of toxic secretion of certain fungi, accordingly merits the designation of ‘Antibody”’.

More evidence was given by DEMETER (1923) in a study of Vinca mycorrhizae: The arbuscles are tree-like branched hyphae, exhibiting in their end branches in young stages little granules arranged in nubelae and strongly staining with haematoxylin. From their origin these nubelae are protein precipitates which have arisen thus: The arbuscle-tips, as a result of action of free H-Ion in the Cell-Sap, burst and emptied their content into the host-cell. It is possibie to form these “plasmoptyse” in the endophyte in pure culture, the action occurring at an optimum acidity of 0,025 N HCI. On the basis of this observation, the name of ‘plasmoptyse’ was chosen. The sporangioles are the structur less residue of the arbuscles which have been made harmless by plasmoptyse, the residue being finally resorbed.

The intracellular arbuscles, said ENDRIQKEIT (1937), cannot be interpreted as assimilatory organs, since they are digested as they are formed and show no indication of hyphal development from their terminal branches, but rather as proliferations induced by the growth- promoting stimuli of the cell-sap. (The expression “growth promoting” used here is perhaps unfortunat,e, since the arbuscle is plasmolyzed.)

180 ARTHUR PIEJMON KELI.EP

“I think with NICOLAS”, said EMBERQER (1937), “that the (mycorrhizal) association is simply accid ntal; and that localization of infection is conditioned by differences of osmotic pressure” So also VUILLEMIN, reviewing GALLAUD’S 1905 paper: The habitual destiny of the arbuscles may be considered less a characteristic production of the endophyte, and more the result of the reaction of the host-cells to invasion by a foreign body.

Fungal digestion in Orchis occurs chiefly from autumn into winter, penetration of the fungus being accompanied by solution of starch in the plant cells. The fungus follows the concentration gradient. (FUCHS, 1924).

Mycorrhizal cells of roots of some investigated orchids, after treatment with concentrated solutions, become plasmolyzed. But those cells which are filled with living hyphae are signi- ficantly resistant to plasmolysis, and were formerly held to be un-plasmolyzable ; yet after appropriate treatment it was possible to plasmolyze these cells. All mycorrhizal cells res- pond to the stimulus of plasmolysis with marked plasma-systrophy. (GERM, 1934).

And so we come to MACDOUOAL (1943), who has come closest of all to giving us an inter- pretation of mycotrophy. In working on Montery pine, he found that phosphorus is most abundant in root-tip and stele and becomes relatively less prevalent as the cell ages and develops large vacuoles. Counterstained with acid fuchsin, a vivid contrast develops between red mitochondria (coated with lipids) and purple plastids, which contain starch grains. Pericycle and endodermis separate the stele which is rich in phosphorus-linkages, and the cortex which is rich in catechol and catechol linkages: fungi infect only the cortex. Phos- phorus must be taken from the soil by mycorrhizal fungi, and this phenomenon is indicated by growth of excised mycorrhizal roots in soil. Hyphae of the mycorrhizal fungi appear to contain phosphorus-complexes, but i t is not clear whether these compounds are synthesized from elementary constituents or from molecular structures. Hyphae penetrate where there is little phosphorus-complex t o act as dehydrogenase. ‘*Such a difference should play a role in controlling selective permeability : Anions, with their negative charge, should be carried from the site of higher activity to that of the lower. The tissues of the stele, from their meristematic stage, maintain a low oxidase level, by retaining a high level of phosphoric complexes, acting as dehydrogenases. This condition enables them t o trap such anions as ‘H,PO, or” HPO, ... Here lies perhaps the essential difference between suffering from parasitism or indulging in symbiosis: in the first case the infected tissues Gould release their phosphoric anions to the parasitic fungus, which is able to maintain a low level of oxidase activity; in the case of mycorrhizal symbiosis, the fungal partner, while infecting the cortical tissues, is never allowed into the stele, wherein there prevails a low level of oxidase and a high level of phosphoric complexes.“

With all this evidence at hand, one might venture to believe that localization and control of the endophyte in mycotrophic organs by ionic forces is not altogether a chimerical fancy.

L i t e r a t u r e c i t a t i o n s BERNARD, N., 1911. Sur la fonction fungicide des bulbes d’ophrydees. Anii. d. Sci. nat. Bot.

RURQES, A.. 1936. On the significance of myrorrhiza. New Phytol. 36, 117-131. -, 1937. The defensive mechanism in orchid mycorrhiza. New Phytol. 28 (3), 273-283. DEMETER, K., 1923. Uber ,.Plasmoptysen“-Mykorrhiza. Flora. 116, 405 -456. EMBEROER, I., cf. p. 166. ENDRIQKEIT, A., 1937. Beitrage zum ernahrungsphysiologischen Problem der Mykorrhiza

unter besonderer Beriicksichtigung des Baues und der Funktion der Wurzel- und Pib- membranen. Rot. Arch. 39, 1-86.

Fucrrs, A., 1924. Aus der Monographie der Orchis Traunsteinrri SAUT. V. Die Pilzvcrdauung der Orchideen. Bot. Archiv. 6, 193-206.

GALLAUD, I., 1905. Etudes sur les mycorhizes endotrophes. Rev. g6n. Bot. 17. 5-48. GERM, H., 1934. Uber den Mykorrhiza-Protoplasten der Orchideen. Ber. Dtsch. bot. Ges. 5?.

MACDOUOAL, D. T., 1943. Study of symbiosis of Monterey pine with fungi. Yearb. Amer.

MAGROU, J., 1921. Symbiose et tubkrisation. Ann. d. Sci. nat. Bot. 10 me. sCr. 3, 181 -296. NOB~COURT, P., 1923. Sur la production d’anticorps par les tubercles les Ophrydees. Cornp.

VUILLEMIN, P., 1905. Review in Bot. Cantralbl. 96, 117-119.

9me. sCr. 14, 221-234.

26-42.

Phil. SOC. 170-174.

rend. Acad. Sci., Paris. 177, 1065-1057.

sammelbericht. localization of the endophyte in mycotrophic organs

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