Phytopath. Z., 71, 1—20 (1971)© 1971 Verlag Paul Parey, Berlin und HamburR

Department of Botany, University of Delhi, Delhi 7, India

The Structure of Launaea asplenifolia infected withPuccinia butleri with a Discussion on the Recurrence of the Rust

By

HARDEV SINGH and K. K. PUSHPAVATHY

With 14 figures

Received April 2, 1970

Puccinia butleri, an autoecious macrocyclic rust, alters the growth patternof its host Launaea asplenifolia (Compositae). The effect is brought about onlyby the aecial stages. The bud on the root-stock which usually produces a stemwith radical leaves forms an elongated axis bearing cauline foliage after infec-tion. In the present paper the morphological and anatomical characteristics ofdiseased plants and the seasonal development of the rust mycelium are studied.

Material and Methods

Unifected and infected plants of Launaea asplenifolia werc collected from theJamuna Banks, New Delhi, and the Delhi University Campus. Part of the material was alsoobtained from plants grown from root-scodis in the departmental botanical garden..Formalin-acetic-alcohol (50% ethyl alcohol 90 ml; formalin 5 ml; glacial acetic acid 5 ml)and Craf — equal quantities of solution A {diromic acid 1 g; glacial acetic acid 7 ml; waterto 100 ml) and solution B (formalin 30 ml; water 70 ml) mixed just before using — wereemployed as fixatives. The material fixed in FAA was stored in 70 % ethyl alcohol whereasthat in Craf was washed in running tap water for about 24 h before passing through thedehydrating series. The material was dehydrated in ethyl alcohol-xylol series, embedded inparaffin wax (M.P. 56—58 °C) and longitudinal and transverse sections were cut at 6—15 «.Safranin-fast green and Heidenhain's hematoxylin with either safranin or fast green as acounterstain were used for staining the sections with satisfactory results.

External Morphology

Uninfected Plant

In India, Launaea asplenifolia (Compositae) occurs in the Indo-GangeticPlain, Assam and Andaman Islands (HOOKER 1882, DUTHIE 1960). In Delhi, it

Phytopath. Z., Bd.71, Hefi 1

SINGH and PUSHPAVATHV

Fig. 1. Morphology of uninft.'ctfc'd plant. A Plant with abuveground and underground parts(vertical root-scock and Iiori7.ont3l root), X 0.35. B Parts of root-stocks with buds andlateral roots, X 1.33. C As in B; aerial shoot decayed and enlarged buds; X 2. D Plantwith two brandied flowering shoots bearing mature capitula; X 0.33. E L of capitulum;all flowers are ray florets; X 1.73. F Floret showing pappus, corolla, staminal column,

stigma and ovary; X 2.Abbreviations (valid for all figures): ae aecium, as aerial shoot, b bud, br bract, ck cork,das decayed aerial shoot, dv distorted vessel, es erect shoot, h haustorlum, hy hypha,If lateral root, It laticiferous tube, nh host cell nucleus, o ovary, p pappus, r root,rs root-stodi, s septum, sc staminal column, sh sheath, sp spcrmogonium, te telium,

ur uredium, vb vascular bundle, L longisection, T transection

grows during September to June in moist shady places, lawns and along thebanks of the Jamuna river. It is a herbaceous weed with extensive and perennialunderground parts (roots and root-stocks) and ephemeral aboveground parts.Soon after the rains (in the beginning of September) the buds on the root and

The Structure of Launaea asplenifoUa infected with Puccinia butleri

root-stodc develop into shoots which come above the surface of the soil and beara rosette of 8—12 leaves. The rosettes remain vegetative during the first grow-ing season (September—June). The leaves die off during summer and the tip ofthe root-stock produces another rosette of leaves during September. The rosettesof the second year remain vegetative for the next four months and begin toflower in January and continue till June. Soon after flowering the aerial partsincluding the tip of the root-stock die off. Thus the rosette has a biennial type ofgrowth and development.

The main root system is buried very deep in the soil and grows horizontallyand obliquely (fig. 1 A). The main root bears a few lateral roots and numerousadventitious buds at random. Some of tbe buds (3 or 4 per main root) grow intovertical root-stocks which bear the aerial shoots. The root-stock Is 15—20 cmlong and bears numerous alternately arranged buds at short intervals (figs. 1 B,C; 7C). Whenever the aerial stem is damaged, these buds sprout and producenew root-stocks and aerial shoots. A few unbranched, thin and fragile rootsarise from the root-stock (fig. 1 B). They also bear buds.

Tbe aerial stem whidi bears a rosette of leaves has indistinct internodes.However, when the plants grow in a dense shade, the rosette habit is disturbedand the stem tends to grow taller thus exhibiting short internodes.

The leaves are purplish-red to begin with but they soon turn green. Theyare alternate, simple, thin, sessile, narrowly obovate, sinuately lobed and havea tootbed margin. The young leaves are pubescent but the hairs are evanescent.All the parts of the plant contain a milk-white latex.

A rosette of the second year bears 3—5 flowering axes eadi one arising inthe axil of a leaf. The flowering shoot is brandied and bears capitula on thesecondary and tertiary axes in a cymose manner (fig. 1 D). The secondary andthe subsequent brandies arise in the axil of small bracts. Tbe capitula arepeduncled and each capitulum is surrounded by 3 or 4 whorls of persistentinvolucral bracts. In a capitulum there are about 30 flowers and all of them areat the same stage of development (fig. 1 E). They are ligulate and bisexual(fig. 1 F). The calyx is represented by numerous persistent stiff bairs, the pappus(fig. 1 F). The corolla is gamopetalous, ligulate and five-lobed at the top. Thereare five, epipetalous and syngenecious stamens with distinct and free filaments.In the young flowers, the anthers envelop the style and stigma and form atubular structure. The gynoecium is bicarpellary with a unilocular inferiorovary containing an anatropous ovule. The style bears two stigmas althoughoccasionally three have also been observed. At anthesis the two stigmas emergeout of the staminal tube and become reflexed (fig. 1 F).

Infected Plants

The newly infected plant (beginning of October) is quite similar to theuninfected one but 2—8 buds on the root-stodt of the infected plant appearwell-developed (fig. 2 A). These are spongy, swollen and grow very rapidlyinto erect shoots (fig. 2B) which possess long internodes. The shoots are pro-

4 SINGH and PUSHPAVATHY

f usely branched at the base andseveral sudi shoots impart awitches' broom appearance tothe infected plants {fig. 2C).Occasionally these shoots aredistorted (fig. 2D). Numerousspermogonia and aecia are pre-sent on the erect shoots (fig.3 A). The leaves borne onthese shoots are pinkish, small,narrow and thick. In Maysome of the erect shoots withspermogonia and aecia evenproduce capitula. The latterdo not develop fully and theyopen only imperfectly (fig.3 B). A number of spermogoniaand aecia are formed on theinflorescence axes, involucralbracts, corolla tubes, stamensand pistils (fig. 3 C to E).

In many infected plantsthe lower leaves form a rosetteand resemble the leaves of theuninfected plants whereas theupper leaves which show sperm-ogonia and aecia are borne ona slightly elongated axis. Lateron the stem elongates furtherand the infected plant becomeserect. In this instance only oneerect shoot is formed.

From the end of January till late in March, the infection spreads by aecio-spores and the newly infected plants develop only uredosori on their leaves.During this period, the rosette of leaves which were associated with the erectinfected shoots may also become infected and show the presence of uredosori.The uredosori are found scattered on the abaxial as well as on the adaxia!surfaces of the leaves (fig. 4 A). As the sori grow older, a ring of secondarysori arises around the primary sorus (fig. 4B). Rarely a few small aecia maybe formed around a primary uredosorus (fig. 4C).

Towards the last week of March, the plants which showed only uredia startbearing telia. The latter are also distributed on both the surfaces of the leaves(fig. 4 b , E). Plants bearing only uredia and telia are similar to the uninfectedplants in having a rosette of leaves and they even bloom normally despite heavyinfection on the inflorescence axes.

Fig. 2. Morphology ot intociL-d plant. Awith a rosette of uninfected leaves and three in-fected, swollen and sprouting buds; X 0.66. B Laterstage in the development of buds forming erectshoots; thf infected plant bears a rosette of un-infected !e:ives; X 0.66. C Diseased plant (spcrmo-gonial and aecial stages) with elongated internodcsand repeated branching of shoots; X 0.33. D In-fected plant (bearing sp and ae) with distorted erect

shoots; X 0.50

T h e S t r L i c t u r c of Launaea asplertifoiia i n f e c t e d w i t h Pticci>ii.i h»t!eri

Fig. 3. Morphology of infected plant. A Lower surface of UMVI"- \\ iili .iccia; X 2.39.B Capitula from healthy (left) and diseased (right) phints; infected inflorescences incom-pletely opened, with aecia on axis and involucral bracts; X 0.6. C Part of inflorescenceaxis with aecia; X 1.5. D Upper part of pistil with spermogonia on the style; X 63.

E T of anther with aecium; X 203

At the end of the active season of infection (April—May), most of therosette plants which had only uredia and telia produce erect shoots bearingspermogonia and aecia. These erect shoots on the plant may arise:

a) from the buds on the root-stock (fig. 5);b) on the aerial stem in the axil of leaves of the rosette;c) by the sprouting of the shoot apex itself (fig. 6 A);d) in the axil of the bracts on the inflorescence axis (fig. 6B).

In the first three instances, when the shoots are young, spermogonia andaecia occur on the vegetative leaves. However, later when they produce capi-tula, the floral parts also become infected and bear spermogonia and aecia. Ingeneral, the development of the capitula borne by these erect shoots is arrested-

Thus three types of infected plants are found: (a) erect plants which showonly spermogonia and aecia. These plants are always associated with a com-plete rosette or a few uninfected leaves at the base. They flower only rarely,(b) Rosette plants with uredia and telia. They bear normal-looking flowers.

SINGH and PUSHPAVATHY

Fig. 4. Morphology of infected plant. A Infected plant (rosette habit) with scatteredurcdia on adaxial leaf surfaces; X 0.66. B Adaxial leaf surface with urcdia in rings; X 1.32.C Abaxial leaf surface with aecia in the vicinity of a uredium; X 4.62. D Heavily infectedplant with scattered telia on the abaxial leaf surface; X 0.66. E Infected leaf with telia

in rings on the lower surface; X 2

(c) Plants which are rosettes and bear erect shoots too; the rosette of leavesshows urcdia and telia while the leaves on the erect shoots develop spermogonia

and aecia. There are normal aswell as underdeveloped capitulaon different parts of the sameinflorescence axis.

Towards the end of May orearly June, the aerial parts ofinfected plants dry away where-as normal plants continue toflourish for another month or so.

Fig. 5. Infected plant with uredia, telia(right) and aecia (left) on different

branches. X 0.56

The Structure of Launaea asplenifolia infected with Puccinia butleri

8Fig. 6. Morphology of uninfected plant. A Infected plant with a leaf rosette bearing urediaand telia and an erect shoot with aecia; X 0.73. B Upper part of infected inflorescence with

aecia on erect shoot and telia on the bract; X 0.73

Anatomy of the Uninfected Plant

Root

A transverse section of the root which has undergone secondary growth shows 3 or4 layers of cork, 10—15 layers of cortex and a central core of vascular system (fig. 7 A).The cortex is parendiymatous and shows many intercellular spaces. There are several latici-ferous tubes in the cortex and secondary phloem. The primary xylem is diarch. Thesecondary xylem contains very conspicuous vessels. The vascular rays are 6—10 layers wide.

Root-Stock

A transverse section of the young root-stock shows an epidermis, a parendiymatouscortex of 7 or 8 layers of cells, a ring of 5—7 collateral vascular bundles and a parendiy-matous pith which is 5 or 6 cells wide. The cells of the epidermis degenerate soon. Initialsof laticiferous tubes can be marked out in the cortex. The cortex also shows many inter-cellular spaces. The medullary rays are 3 or 4 cells in width.

Following secondary growth, 4—6 layers of cork cells are formed (fig. 7B). Thelaticiferous tubes become conspicuous in the cortex (fig. 7C, D) as also in the secondaryphloem. The vascular rays are 3—8 cells in width.

Aerial stem

In transverse section, a stem with the primary structure shows a thinly cutinizedepidermis covering the cortex, stele and pith (fig. 8 A, B). The epidermal cells are rectangularand contain a few granules of tannin. The cortex consists of 6 or 7 layers of parendiymatouscells whidi show a few intercellular spaces. There are many collateral and open vascular

SINGH and PUSHPAVATHY

Fig. 7. Anatoin> ot unuili;ctcd plant. A T ot root sbowing a central core ot xylcm envelopedby cortex and cork cells and part of a lateral bud; X 20.5. B T of root-stodi; X 19. C L ofroot-stodt bearing a bud, with distinct laticifers; X 20.5. D L of a laticiferous tube from

root-stock cortex; X 210

bundles whidi are arranged in tbe form of a ring. The stem being condensed, leaf tracebundles arc encountered in most transverse sections. The medullary rays are 3—6 cellsin width. The pith is composed of 10—15 layers of parendiymatous cells wbich show afew intercellular spaces.

The aerial stem which has undergone secondary growth is covered by 4—6 layers ofcork cells (fig. 8 C. D). Tbe cortical cells are tangentially elongated. The secondary phloemand cortex have numerous mature laticiferous tubes. The vascular rays are 3—S cells inwidth. The pith cells enlarge during secondary growth. Some of the pith cells associatedwith the leaf trace undergo an occasional division.

Leaf

The very young leaves are pubescent; the hairs are uniseriate and have a swollenapical cell. The mature leaves, bowever, are devoid of tridiomes, A vertical section of themature leaf shows an upper and lower epidermis, mesophyll and vascular system. The cellscomprising the epidermis are rectangular to squarish. The stomata are present on bothsurfaces of tbe leaf. The mesophyll comprises thin-walled, rounded and compactly arrangedcells which arc not differentiated into palisade and spongy parenchyma. Tbe lamina hasa number of collateral vascular bundles which have a cap of collenchyma on either side.

FlowerT

The youngest anther available in our material showed four lobes in a transversesection. Each lobe contained a few primary sporogenous cells (2—5 in t.s.) and tbe developingwall layers. The fully developed anther shows four wall layers. The cells of the endothecium

The Structure of LaHnaea asplenifoUa infected with Puccinia butleri

Fig. 8. Anatomy of uninfected plant. A T of young aerial stem with wide pith; X 40.B Part from A to show the structure of a young vascular bundle; X 132. C T of old aerialstem with numerous Iaticiferous tubes in phloem and cortex and leaf bases around the stem;X 31, D Portion from C to show the structure of vascular tissue with distinct Iaticiferous

tubes; X 95

do not show the characteristic thidcenmgs. The innermost wall layer forms the tapetum.At the sporogenous cell stage the tapctal cells are uninucleate but by the time the anthershows microspores the tapetal cells become multinucleate and show dense cytoplasm. Thosporogenous cells give rise to the microspore mother cell. Meiosis I and II lead to thelormation of tetrahcdral or decussate tetrads. The microspores in the tetrads soon separateIrom each other and become thick-walled. The exine shows radial markings and the intinebears spiny projections.

The ovule is anatropous, unitcgminal and tenuinucellate. The integument is verymassive and well differentiated at the megaspore mother cell stage. The megaspore mothercell forms a linear tetrad of megaspores, of whidi the dialazal one functions. However, anovule showed the two middle megaspores enlarging. The mature embryo sac is 8-nucleate.The synergids arc long; the polar nuclei fuse very early and the three antipodals areephemeral. The egg nucleus lies at the apex of the egg-cell and the primary endospermnucleus is placed just below the egg. There is a well-developed endothellum around theembryo sac.

10 SINGH and PUSHPAVATHY

Anatomy of the infected Plant

Root

The roots of the infected plants have never been found to show any hyphaeand anatomically they are similar to the roots of the uninfected plants.

Root-stock

The portion of the root-stock whidi lies deep in the soil is free of hyphaeand is similar to the uninfected root-stock in its internal structure. The hyphaepenetrate the root-stock only up to a few centimeters below the soil level. Thefollowing description applies only to the infected portion of the root-stock.

a) Plants bearing spermogonia aad aecia

In the heavily infected root-stodt, there is a hypertrophy and hyperplasiaof the cortical cells, and the cortex comprises 10—15 layers of cells. The numberof tracheids is large and that of the vessels is very small. The cells of the pith arealso hypertrophied. Some of the pith cells become thitk-walled and pitted andshow haustoria (fig. 10 A).

Fig. 9. Anatomy of plant with spermogonia and aecia. A T of root-stock with hyphae; X 26.B Portion marked B in A with intercellular hyphae and a few haustoria; X 172

Intercellular hyphae are present in the cortex, vascular tissue and the pith(fig. 9 A, B). Several hyphae are also seen in the lumen of the xylem vessels(fig. 10 B—D) and tradieids. They are brandied, septate and dikaryotic ormonokaryotic. The hyphae enter the xylem vessels through the pits. The hyphaealso traverse inbetween the tradieicis and vessels and thereby cause their distor-

The Structure of Launaea asplenifolia infected with Puccinia buileri nFig, to. Anatomy of plant withspermogonia and eacia. A Root-stock pith with two thick walled,pitted idioblastic cells with hau-storia; X 349. B L of root-stockwith a septate and dikaryotichyphae in xylem; X 446. C Tof xylem vessel with brandiedi.septate hyphae; X 1000. D Di-storted vessels in the root-stock;

X262

tions (fig. 10 D). The hyphaefrequently send hyphae-likehaustoria inside the pithcells (fig. 11 A—C) and cor-tical cells. The haustoriumcomprises unbranched orvariously branched proces-ses joined to a common basalpart. Sometimes, the haus-torium surrounds the nucleusof the host cell (fig. I IB) .Two or three nuclei may bepresent in eadi haustorium(fig. 11 A, C).

b) Plants bearing uredia and telia

Anatomically the root-stock of these plants is identical to the uninfectedones. The hyphae do not seem to penetrate in it.

c) Plants bearing all types of sori

The root-stodts are similar to those of the plants bearing only spermogoniaand aecia. Numerous variously brandied intercellular monokaryotic and di-karyotic hyphae are present in the cortex, vascular rays and the pith. Occa-sionally structures resembling internal spermogonia are seen in the vascularregion (fig. 12 B). Hyphae are seen inside xylem vessels (fig. 12 A). Branchedhaustoria are also common.

Aerial stem

a) Plants with spermogonia and aeciaThe stem shows a tanniniferous epidermis which has a few stomata. The

cortex is composed of 6—8 layers of parendiymatous cells which have manyintercellular spaces. In contrast to the uninfected stems, in these stems, thenumber of vascular bundles is mudi larger (as many as 14 bundles have beenobserved; fig. 13 A). The vascular bundles are open and collateral. The medul-lary rays are 4 or 5 layers wide. Unlike the uninfected stem, occasionally, alayer of scelerendiyma can be seen towards the xylem of the vascular bundles

SINGH and PUSHPAVATHY

Fig. 11. Anatomy of in-fected plant. A, B Pithcelk from root-stodt (plantwith sp and ac) showingbranched haustoria (bi-nucleaie in A) and host-ccllnuclei; in B, host cell nu-cleus partially encircled byhaustorial lobes and poor inchromarin; X 465. C Un-brandied haustorium in apith cell, with three nuclei;X 465. D Cortical cellfrom erect aerial stem{plants with sp and ae) witha haustorium and a distinctsheath around it; host nu-cleus abnormally elongated;X 465; E Cell from a leafbearing ur with a coiledhaustorium with a sheathat the base; host nucleus is

away from it; X 465

of the infected stem. The piih is parendiymatous and has many conspicuousintercellular spaces. As compared to the uninfected stems, there is very littlesecondary growth in these stems. The phloem shows laticiferous tubes.

A few monokaryotic and dikaryotic intercellular hyphae are present mthe cortex, phloem, pith, and also the shoot apex of the host. These hyphae send

Fig. 12. Anatomy of plant with alltypes of sori. A Dikaryotic hyphain xylem vessel; X 571. B Part ofroot-stock xylem with a young in-ternal sp and a hypha in the vessel;

X200

The Structure of Launaea asplenifolia infected with Puccinia butleri 13

•aoMFig. 13. Anatomy of plant with spermogonia and aecia. A 1 m L . I ^ I item with wide pith

and many vascular bundles; X 27.5, B T of erect stem with an open aecium; X 81

variously branched haustoria in the host cells. Occasionally, the haustoriasurround the nucleus of the host cell and these nuclei sometimes become hyper-trophied and look lens-like (fig. 11 D). The mature haustoria have sheathsaround them and are enucleate (fig. 11 D). Hyphae also occur inside xylemvessels. At times, spermogonia and aecia develop on these stems (fig. 13 B).

b) Plants with uredia and telia only

Anatomically the structure of the aerial stem of these plants is identicalwith that of the uninfected aerial stem whidi has undergone secondary growth.The stem is devoid of hyphae.

c) Plants bearing all types of soriOnly the plants in which the shoot (bearing spermogonia and aecia) arise

in the axil of leaves on the aerial shoot, have been studied. These consist of athick main stem and slender pinkish side shoots which grow erect. The anatomyof these shoots is identical with the shoots producing spermogonia and aecia.The shoot apex shows many dikaryotic hyphae and the nuclei in some of thehost cells become abnormally elongated.

The main thick stem has dikaryotic and intercellular hyphae in the cortex,pith and the xylem vessels. Richly brandied haustoria are seen in the corticalcells. MOfCgover, patdies of monokaryotic hyphae are seen in the intercellularspaces of .tlj ^v ijcular rays. Occasionally teliospores are seen embedded in thecells of cpft i tof j:he main stem or in leaf bases. They occur either singly or insmall groups,Sg^^ne of the pith cells become pitted.

, ' • Lear

i a) Leaf bearing spermogonia and aecia

The mesophyll cells show hypertrophy and hyperplasia, thus there is anincrease in thidkness of the diseased leaves. The intercellular spaces are packed

14 SINGH and PUSHPAVATHY

with hyphae. Branched haustoria are present in the cells of epidermis and meso-phyli. Hyphae occur inside the xylem vessels too. Spermogonia and aecia arepresent on both surfaces of the leaf as well as of the midrib.

I b) Leaf possessing uredia and telia

A transverse section of the leaf shows the following differences as com-pared to the uninfected one. The midrib is somewhat flattened. There are manyhyphae in the substomatal cavities. Variously branched hyphae are also presentin abundance in the intercellular spaces. Binucleate unbranched or branchedhaustoria are of common occurrence in the cells of epidermis and the mesophyll.Sometimes the haustorium is coiled like a spring (fig. 11 E). The lamina bearsuredosori or teliosori on either side.

Flower

a) Plants with uredia and telia

The development of microspore and the structure of the ovule appears tobe similar to those in the uninfected flowers. The formation of megasporemother cell, linear tetrad and 4-nucleate embryo sac have been observed in theseflowers. I

b) Plants with spermogonia and aecia

The hyphae occur in the tissue of the infected flowers and spermogonia andaecia are found on the involucral bract, corolla tube, stamen and pistil. Thestamens whidi showed hyphae in the connective had normal-looking micro-spore mother cells. The development of spermogonia or aecia on the corollatube or the connective of the anther did not cause any visible change in thestructure of the microspores; they were similar to those found in the uninfectedflowers. However, the anther appeared rather distorted.

The megaspore mother cell as also the embryo sac developed despite thepresence of hyphae in the ovary wall of the floret. Tn the infected flowers, themycelium occurs especially abundantly either at the base of the ovary or in theintegument or the ovary wall. The ovary and ovule do not go through post-fertilization development.

Seasonal Growth and Location of the Mycelium

The rosettes arising from root-stocks (which bore infected plants in theprevious season) in the new season (September—October) do not show anysymptoms of rust infection. An anatomical study of the shoot apex, leaf, aerialstem and root-stock of these healthy-looking plants collected in September didnot reveal any mycelium. However, in October, the shoot apex of the plantsshowed hyphae (fig. 14 A, B). Later (November—Eebruary), when the plantsbear spermogonia and aecia on their leaves, the root-stock portion just belowthe soil surface harboured mono- as well as dikaryotic hyphae (fig. 14 C). Evenat this time, the deep-lying root-stotk of the infected plant was devoid of

The Structure of Launaea asplenifolia infected with Puccinia butleri 15

hyphae (fig. 14D). This shows that in the beginning the plants are uninfected;later the shoot apex becomes infected and eventually the hyphae grow from theaerial to the underground part i. e. the root-stodt. During November—Maywhen the plants are severely infected aerially, an abundance of mycelium is seenin stem and leaves (fig. 14 E—H) and also a portion of root-stock a few centi-meters below the soil surface but there are no hyphae in root-stodt lying deepin the soil or roots, the only two parts which are perennial. After the death of

Fig. 14. Seasonal distribution of the mycelium in the host. A L of shoot tip (early October)of newly infected plant with a few hyphae below the apical dome; X 31, B As A, advancedstage (late October) with abundant hyphae in the tip; X 31. C T of root-stock (February,plant with sp and ae) with hyphae in cortex and pith; X 20. D T of deep-lying root-stock(February, plant with sp and ae) without hyphae; X 20. E T of erect shoot (January, plant•with sp and ae) with hyphae in cortex and pith; X 20. F T of leaf with aecia on bothsurfaces (January) and numerous hyphae; X 10. G Leaf section with uredosorus on uppersurface (February) with a few hyphae in the lamina; X 10. H Leaf section with telia on

both surfaces (April) and hyphae adjacent to the sori; X 20

16 SINGH and PUSHPAVATHY

the aerial parts of the severely infected plants (June—July) the dug out root-stocks and roots showed no mycelium. This indicates that the mycelium does notperennate in the deep-lying portions of root-stock or roots.

Discussion '

DUTHIE (1960; see also DALELA 1963) mentioned that Launaea aspleni-folia is biennial or perennial. The present study shows that the plant is peren-nial but each rosette is biennial i.e. the latter is vegetative in the first season(the leaves drying up in summer and new leaves appearing on the same shoottip during September—October) and flowers in the second season after whidithe apex of the rosette and the short aerial stem die. The deep-lying root-stodito whieh the rosette was attadied remains alive and fresh rosettes are borne on itin the next growing season. DUTHIE (1960) wrote that in L. asplenifolia theflowering shoots arise from the roots but according to the present observationsthe flowering shoots arise in the axil of leaves present on the condensed aerialstem (fig. 1 D).

According to BUTLER and JONES (1949) the formation of erect shootscaused by rust infection of L. asplenifolia is an example of an organoid gall(characterized by changes in the morphological plan of shoots or other affectedorgans). Our anatomical studies show that the infected plants do not vary mudiinternally and thus support BUTLER'S and JONES' view. However, two majordifferences (the absence of cork formation and secondary growth) have beenfound in the organoid galls of L. asplenifolia.

In the erect .shoots of L. asplenifolia we observed lens-like nuclei whidiwere 2—3 times bigger than in uninfected cells. This corresponds to earlierobservations with other rusts (ALLEN 1923, AKAI 1951, WHITNEY et al. 1962,SHA'ff 1967).

Usually the hyphae of rusts traverse the intercellular spaces of the softtissues of the host. The mycelium of certain rusts, however, enters the lumen ofxylem vessels and tradieids (PADY 1935, RICE 1945, ZIMMER 1955, PETERSONand ScHURTLEFF 1965). In our study too an abundance of hyphae has been foundin the vessels of the infected root-stock of plants bearing spermogonia and aecia.The growth of hyphae between the walls of the adjacent vessels results in adistortion of the latter. The erect shoots of infected plants dry away soonerthan the healthy ones; this may be due to the hyphae in the vessels whidi mayhinder the transport of water.

In some fungi like Cronartium the haustoria have a definite shape whichmay be used in species identification (COLLEY 1918). The haustoria of P. hutleriare unspecialized and hypha-like as in certain other species (HUNTER 1948).According to ALLEN (1930) "it is probable, however, that all these intracellulargrowths whether haustorial in form or intermediate between that and ordinaryhyphae are haustorial in function".

The Structure of Launaea asplenifolia infected with Puccinia hutleri IJ

Since ROSEN'S observations (1893) the haustoria of many rusts have beenshown to elongate towards the nucleus of the host cell and envelop it partiallyor wholly (AKAI 1951). SAVILE (1939) mentioned that the haustorium ofUromyces fahae closely invests and distorts the host nucleus; some authors havespoken of a degeneration of the nucleus when in association with the hausto-rium {Moss 1926). Generally the infected cells have a hypodiromatic nucleus(KiJsTER 1930) but in Clematis paniculata infected with Aecidium clematidis,it is hyperchromatic. The nuclei of infected Launaea cells are typically hypo-chromatic and stain faintly.

MEHTA (1918) found mycelium in the root-stodt of Launaea plants bear-ing aecia and thought that it might play a ro!e in the recurrence of the disease.He did not indicate the depth to which the mycelium had penetrated into theroot-stock. We found that during November—May when the plants are seve-rely infected aerially the mycelium occurs in root-stocks which lie only a fewcm below soil but never in the deep-lying root-stock portions or roots. Duringsummer, when the aerial parts of the plant dry away, no mycelium was foundin the root-stock. Therefore the mycelium does not seem to be perennial. Earlyin the season a rosette free from sori develops; later erect shoots bearing sper-mogonia and aecia are formed. The transplantation experiments of MEHTA(1918) do not give conclusive proof of a perennial mycelium and his infectedplants might have been the result of fresh infection.

We studied the distribution of the mycelium during different seasons andfound that in plants with uredia and telia dikaryotic mycelium occurs only inthe leaves. In root-stock and aerial shoots of plants bearing aecial stages wefound mono- and dikaryotic mycelium as observed by DALELA (1963). At thebeginning (October) hyphae are present only in the aerial parts and only whenthe aecia mature (November—February) the hyphae are seen in abundance inthe root-stock. Thus it seems that the aerial parts become infected first and thehyphae grow later to the underground parts. In the absence of detailed ana-tomical studies of plants in all seasons DALELA (1963) came to the erroneousconclusion that the mycelium perennates in the root-stock.

I Summary

Launaea asplenifolia has a perennial root-stotk and a biennial rosettewhich produces capitula in the second season. Puccinia butleri infects the plantduring October—May. The freshly infected plants show a rosette as the healthyplants but the buds on the root-stock grow into elongated shoots which oftenbecome profusely branched as witdies' brooms; their leaves develop spermo-gonia and aecia. In January—March plants are infected with aeciospores anddevelop uredosori; towards the end of March telia are formed. Like healthyindividuals the plants bearing uredia and telia remain rosettes. During April toMay these plants develop erect shoots in the axil of leaves of the rosette, in theaxil of bracts on the inflorescence axis and from the buds on the root-stock. Thecapitula produced by the erect shoots develop numerous spermogonia and aecia.

Phyiopiih. Z., Bd. 71, Heft 1

SINGH and PUSHPAVATHY

Three types of plants are thus recognizable: plants with spermogonia and aecia,plants with uredia and telia and plants producing all types of sori. Infectedplants die earlier than healthy ones.

Anatomically the root of the infected plant is similar to that of the healthyplant and has never been found to contain hyphae. The root-stock of plantsbearing uredia and telia only did not contain any hyphae. The root-stock ofplants developing spermogonia and aecia or all four types of sori showed mono-and dikaryotic hyphae penetrating 5—7 cm from soil level into the root-stockand developing branched haustoria in the cortical and medullary cells.

Monokaryotic and dikaryotic hyphae traverse the intercellular spaces andenter xylem vessels through pits. In plants with spermogonia and aecia somevessels are found distorted. The nucleus of infected cells may be abnormallyelongated. Rarely teliospores develop between cells of the aerial stem of plantsbearing all types of sori. The infection does not alter the development of pollenand ovules but infected flowers do not go through post-fertilizatian develop-ment.

Infection is annual and plants are infected afresh every season. In Octoberthe shoot apex or the buds on root-stodts are infected; in October—Novemberthe mycelium grows through the aerial parts into the root-stock but hyphae donot penetrate into the deep lying parts of root-stock and roots.

We are grateful to the authorities of the Department of Botany, University of Delhifor laboratory facilities. One of us (K. K. PUSHPAVATHY) received a Research Fellowship fromthe Council of Scientific and Industrial Researdi, New Delhi.

Zusammenfassung

Der Entwicklungsgang von Puccinia buileri und die Struktur infizicrter Pflanzenvon Launaea asplenifolia

Launaea asplenifolia (Compositae) bildet einen perennierenden Wurzel-stock, aus dem sidi eine zweijahrige Rosette entwickelt. Puccinia butleri infi-ziert die Pflanzen von Oktober bis Mai. Frisch infizierte Pflanzen tragen einenormale Blattrosette; aus den Knospen am Wurzelstodc entwickeln sich ver-langerte.oft hexenbesenartig verzweigte Sprossemit Spermogonien und Acidienauf den Blattern. Im Januar bis Marz infizieren Acidiosporen Pflanzen, dieUredo- und Ende Marz Teleutolager bilden. Diese Pflanzen bleiben rosetten-artig; im April und Mai treiben sie aufrechte Sprosse in den Blattachseln derRosette und der Blutenstandsachse und aus Knospen am Wurzelstod.; die darangebildeten Blutenkopfe tragen Spermogonien und Acidien. Es finden sidi somitPflanzen mit Spermogonien und Acidien, Pfianzen mit Uredo- und Teleuto-lagern und soldie mit alien Sporenlagern.

Die Wurzeln infizierter Pflanzen unterscheiden sich anatomisdh nicht vongesunden und enthalten ebenso wie der Wurzelstock von Pflanzen mit Uredo-und Teleutolagern keine Hyphen. Im Wurzelstock von Pflanzen mit Spermo-gonien und Acidien und soldien mit alien Sporenlagern finden sich mono- und

The Structure of Launaea asplenifolia infected with Puccinia butleri 19

dikaryontische Hyphen bis zu einer Tiefe von 5 bis 7 cm unter dem Boden undverzweigte Haustorien in Rinden- und Markzellen.

Mono- und dikaryontische Hyphen wachsen interzellular und dringendurdi Tijpfel in die Gefafie ein; in Pflanzen mit Spermogonien und Acidiensind die Gefafie zum Teil deformiert. Die Kerne infizierter Zellen sind zum Teilabnorm verlangert. Die Entwicklung von Pollen und Embryosack wird nlehtbeeinflufit, doch erfolgt nach der Befruchtung keine Weiterentwicklung.

Die Pflanzen werden jedes Jahr (im Oktober) durdi Sprofisdieitel oderKnospen frisch infiziert; spater wadist das Myzel in den Wurzelstodi hinunter,dringt jedoch nieht in die tiefer liegenden Teile von Wurzelstod; und Wurzelnein.

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2*

20 SINGH and PUSHPAVATHY, The Structure of Launaea aspUnifoUa

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* Not seen in original.

Authors' addresses: Dr. HARDEV SINCH, Department of Botany, University of Delhi,Delhi-7 (India). Dr. K. K. PUSHPAVATHY, Department of Botany, Gargi College, New Delhi(India).