Group Title: Morphology, development, and ultrastructure of Termitaria snyderi Thaxter /
Title: Morphology, development, and ultrastructure of Termitaria snyderi Thaxter
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Title: Morphology, development, and ultrastructure of Termitaria snyderi Thaxter
Physical Description: 100 leaves : ill. ; 28 cm.
Language: English
Creator: Khan, Saeed-ur-Rehman, 1943-
Publisher: s.n.
Place of Publication: 1973
Copyright Date: 1973
Subject: Pathogenic fungi   ( lcsh )
Termites   ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Thesis: Thesis (Ph. D.)--University of Florida, 1973.
Bibliography: Includes bibliographical references (leaves 94-99).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Saeed-ur-Rehman Khan.
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Bibliographic ID: UF00097581
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000463515
oclc - 37863322
notis - ACM6697


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Oit m -.:.-F ? T'h:, Th.ter




1 7 3


I wish to express my gratitude to Dr. James W. Kimbrough, the

chairman of my supervisory cor.-ittee, for his willing, able and

constant guidance and advice throughout the course of this study.

My special thanks are due to Dr. Henry C. Aldrich, who opened a

new field of ultrastructure to me, for his patient assistance and

his criticism on the preparation of the manuscript.

I also wish to extend my thanks to Drs. Leland Shanor, Dana G.

Griffin, 111, and Pobert E. Stall who worked on my supervisory

committee along with Drs. Kimbrough and Aldrich.

Thanks are accorded to Institute of International Education,

New York, and U. S. State Department for the financial assistance

provided, without which this study could not have been possible.

Finally, I wish to thank my wife, Patricia, for her continuous

encouragement and generous understanding, and for typing and proof-

reading the manuscript.


Acknowledgerents. . . . . . . .

List of illustrations . . . . . .

Abstract. . . . . . . . ...


I The M:orphology and Development.

II Conidiogenesis. . . . .

III Haustorial Mechanis m. . . .

[V Taxonomic Position. . . ...

Bibliography . . . . . . .

Biographical Sketch . . . . . .

. . . . . .

. . . . . .


P.a ge

Plate I. :-.i ... ,.. i.-: habit and habitat. . . 13

Plate II. ";:':-,~: .-*.,i.- light micrographs of the
cros.7 sections of ;porocarp. . . . . .. 15

Plate III. 7 :r-..:rt .:.' r:, haustoriial mother cells. . 1

Plate IV. ;:-':..-' r: ',mtr-, electron micro graph of the
hau;troiumi connected to a h5usEori~ l fniother
cell by a neec .and light micrographs of the
young thallus growing on an antennal segment 19

Plate V. .-;r.,lt. i c .:~- l r. ', electron nicro.-raph of th?
;subh ,enial cell ; and light Ticirograph: of
marginal tip of mtture thalluS ....... 21

Plate VI. ;.:-.-:n:', s,.,;-rt, electron nicrograph of
the subh.,rieniun. ... . . . . . . 23

Plate VII. T-: :.! '-a r- :-.: r conidia and the
phial ide . . . . . . . . . 25

Plate VIII. r- -:"- r:a ~'::,ir', light and electron
nicrow,-ph of the e>cipulum. .... ...... 27

Plate IX. :,r-'":,.-, ., ; developmental stage; . . 29

Plate X. rf'.:t.:r-.: c,:./;.ri, sporodochiun and the
conidi . . . . . . . . . .. 45

Plate (I. .'r-:..e ": ,':l. : electron micrographs of
the conidiophores. .. . . . . .... .. 47

Plate XII. T 'rr-,':..:' -r .1 --:, electron micrographs of
the conidiophores. . . . . . . .. 19

Plate XIII. elrn'.:t.:-J .-: Ar, electron micrcgraph: of
the con diophores. . . . . . . .. 51

Plate XIV. :'i..;:. .'.: : I3. .:', different stages in the
formation of dcjble septum . . . . .. 53

Plate XV. Per'it..l a .,*:,'.;; ', diagranmm tic representation
of the conidiogenesis . . . . . . . 55

Plate XVI. I;rnicitr-, ni,, ri, electron micrographs of
haustorial mother cells. . . . . . . 71

Plate XVII. T-r-.,t..ria ,:'dJeri, electron micrographs of
the hiustorial neck. . . . . .. ..... 73

Plate XVIII. Ter" itzrfa n~,:zi' light and electron microgriphs
of haustoria.. . . . . . . . . 75

Plate .IX ;-m:tar.~ :-*' electron micrographs of
the haustoria. . . . . . . . . . 77

Plate y",. 'ertm"ita'ric s',:.'C, electron micrographs of
haustoria showing mini-microtubules. . . .. .79

Plate XXI. crnit.ri.i r, .-i, electron micrographs of
the haustoria showing lysosones . . . .. 81

Plate .XII. ,'!vti CZ-CllZ.a ,'r.e:, lignt microgriphs of
the cross sections of sporccirp . . . ... 93

bL tract of Disser tt on Present-c to t're Graduate Council
of the ULniers itL of Florida in Partial Ful fiT:mert of the FPuiremr.nts
for tr, Degree of oioc.tor of Philocophy

OF T.:-;:. '._. -~. ": Tha.ter


Saeed-ur-Rehan Khan

June, 1973

ChairmT3an: Dr. James U ?.iia.brough
M'jor D[eparrmrtent: Bltany

!r-.- 'i' a '.z--r:. Theater is an entorr phlilous fungus g"

on the exoskeleton of different species of ter-rites. The fungus

is an ectclparas'to Special :ed thick.-,alled haustorial nm.ther cell;

of the baTal lajyr of the fungal spcrodocnium send raustoria ir-to

the integu,.-int of the The haLstorial peg enters the noet

through the pore canals of the host cuticle.

The haustoria are lobed, uninucleate, surrounded by' a thick

wall ani Separated from the ho:t protcplast by its plasma membrnr.e.

Alon3 with usual organelles the haustoria contain certain *-acuolei

that are: 1sosorTal in nature, and a new organelle, mini-micrDtubule:

that are only 6 to 100 A in diameter and have ;i>. ub-units. The

mini-TicroLutules are found in tun.jies free in tne general cyto-

plasm. between :he cisternae of smooth endoplasmic reticulun,

between ER cisterna and plazT.a rmenbrane or between ER ci5sterna and

the nuclear envelope.

Tre only known means of reproduction is the formation of

conidia that are cut off endogenou;ly :t the phiilide; at the

conidiogerous loci into long collarettes in basipetal succession.

Phialides are closely clustered into a sporodochium. The conidia

are catenate, cylindrical, uninucleate and hyaline.

It is proposed here that the conidia germinate to form a

crust-like primary thallus that later, by the development of

phialides over it, matures into a sporodochium.

The ta.onomic position of T 7niitaria anj its relationship unth

other imperfect fungi are discussed. Within the fungi imperfecti

a new family and a new order, Termitariaceae and Termitariales

respectively, are proposed to accommodate Teirri .a'ia and ,.'2t;rItl .2'..

A new species of ,al.lttcro2.,a based on the slide of Tenit."::riz made

by Tha:ter is also described.



In 1920 Tha.tter described two rnew fungi growing on trhe E .os el.eon

of three species or termites, -- :,t*a :;r-.; ;'L. : .' ; rol ler), ,.

' *; ::, '..5 (E.inls) and .. ,::tr,'T .- ctc-.. (PolaTiren), collected

from Washington, D.C., Sardinia, and Granada rcspectielly. He

proposed a new q tnis w'r~"- .n o ith t.lo spec ies T,J'. :-:,i;ri: and

T'. er-..~- Peichensperger (1921) reported a similar fiun ,us growing

on the e,.oskeleton of ,:; .. ::;t-rr.- -' :r:- (P mt-un), ,.. .. :: ,

(Hagen), C:.- :':-_:.---_ .:, ..- .7 dollarr) ,.--::: ..; ft;:.-T .: i (Fuller)

an v',. z;i'.,; the first three were collected from Brazil and the

others frcrm. the Congo in Africa. He recognized them as I-T:I-.'Ir-

.*tcL r'. Colla (1929) adde3 F. r-, tc .-: 7, '-., : -.' (PR.a tun) and

l'-:, ; s i ;;::;;,: (Silvesteri), both fronr South America, as hosts

of l'-rt c -ic ,Z .-: Tha .ter, and ."z:,:':c r- --r..2 : (MIotschuls' ) ,

A'. ruair,=: (HolrrgrE n) and ,'. :..r.: ::- (Oshi- ) to those of I-:' :.:.'...

co*:'*c.,1 ThaRter. Pickens (1952) mentioned another species ..

p.:c,-:i; :'.3 tut no details were gi'en.

Thaxt:-r considered the furious as an e,.ternil parasite growing

on the surface of the host without any indication of actual penetration

by the parasite through tnr integumrert of the host. He reported that

the fungus did not caus; any irconvieenierc to the insect. However,

Peichensperger (1923) reported defor-mtion of the infected legs and

antennae. Feytaud and Dieu:eide '1927) considered it )n internal

parasite, and showed that the adipose tissue w:as invaded and

altered by the mycelium. The cuticle was penetrated by a root-like

pedicel and in the process the hypodermis .nd cuticle were altered,

cells disappeared near the break, color became abnormal, and large

amoebocytes were found. Heim e. aL. (1951) also thought that this

fungus is an internal parasite which enters the body through

ingestion or licking, invades the fatty tissues, and finally

erupts to the exterior by breaking the exoskeleton of the host.

Thaxter (1920) considered ze -ic. ia a Fungus Imperfectus ano

placed it in family Leptostromaceac, though he noted that its

position there was an isolated one. Heim (1972, personal communication)

thinks this fungus belongs to Tuberculariales of the Fungi Imperfecti.

In and around Gainesville, Florida, the subterranean termites,

Re2tieZeeitez-ie fiavipes and .A. virgainicus have Ter-ritf.-ia sr;yd~r

Thaxter growing on their exasleleton. The rediscovery of Irit-. ri-

has provided an excellent opportunity to study and resolve many

questions about its morphology, development, taxonomy, and mode of


Materials and rMethods

The termites were collected and the studies were done on

naturally growing fungus. Different media here tried but the fungus

could not be cultured. Methods for the studies are described in

detail in the two chapters.


The fungus grous on any of the body parts antennae (Fig. 2),

legs (Fig. 5), mandibles, head, thorax (Fig. 1), and abdomen (Fig. 3) -

the most common and most prominent infection be'ng that of the

abdomen. When growing on an antenna the fungus forms a crust that

completely encircles it (Figs. 2, 15). Mature stages were not

encountered on antennae.

Termitaria does not appear to disturb or irritate the host.

Termites with multiple infections have been found to differ little

in their activity from normal ones. HowE'.er. when the legs rnd

antennae are infected certain d formations result. The number of

segments in artenni decrease (Fig. 2) and leg tecones swollen

(Fig. 5). The atdcomen ray al.o swell but it is hard to detect

because of its globose nature.

During the process of collection, to *,ery irinortant ctbser-

vaticn: were made. The fungus co.:uld be collected year rourd, and

only 6-IO of the tme..ber: of a colony i.ere found infected. Earlier

workers also reported the scarcity of the fungus in infected

colonies (Tha.ter, 1920; eichenrsperger, 1923).


The riature fungus foris a lenticular to hnsterioid sporocarp,

spherical in outline or variously elongated depending upon the

position of its growth. It is closely appressed t. the cuticle of

the termite rad corsists of a ITmainy-l]yered basal pEeudopar'ench.mrtous

suDh.menium from which firmly coherent, simple, parallel conodiophores

arise vertically, forming an even hyTenial surface (Fig. 7). The

whole sporocarp is 70-20 j thick the subhymenium being 11-20 u

and the hyneniuni 50-60 u. The size of the sporocarp ranges from

375 X. 400 1000 u. A thick-walled area divides the h;,enium

into an upper conidium-containing layer and a lower conidium-free

one. The upper zone is 22-25 u thick and the lower 30-25 u.

The peripheral cells are dark and thick walled and form a

well defined, sterile rim or excipulum. The margin in contact with

the substratum is spreading and only one cell thick at the tip

(Fig. 17).

The basal layer is comprised of light-colored cells with dark-

colored, thic-;-walled cells interspersed among them. The structure

and arrangement of these cells can be easily understood by looking

at a younger stage (Figs. 12, 14, 27). They appear pyriform when

viewed from the top, and are often in shiny rows and have a hyeline

spot in the center (Fig. 12). They were considered chlamydospores

by Thaxter (1920), Feytaud and Dieuzeide (1927), and Heim (1952).

This study has shown that the s3-called chlamydospores are actually,

haustorial mother cells (for details please see Chapter III) that

send penetration pegs into the integument through the cuticle.

The hyaline spot in the center in fact marks the position of the

penetration peg. The haustoriun is lobed, uninucleate, the nuclei

lying in the broad base, and has a very thick wall (Figs. 8, 9,

13). The host plasma membrane forms a sheath around the haustorium,

thus at no time is the haustorium in direct contact with the host

cytoplasm. The haustoria could not be traced beyond the basement

membrane, thereby suggesting that the fungus is an ectoparasite.

The haustorial mother cell has been seen both with and without

a nucleus, the forTer situation being rare. It appears as if,after

the haustorirT has reached a certain size, the nucleus of the

haustorial mIc.thrr cell migrates to th7e haustoribm. The haustorial

mother cell is separated from the cell above by a typical ascomycete

septum with Woronin bodies in the vicinity (Fig. 11) and is connected

with the haustorium by a narrow nec! that passes through the cuticle

of the host (Figs. 9, 13).

The subhymenium is comprised of nucleate cells separated from

each other by an ascomycete septum (Figc. 16, 18). These nuclei

are bounded by a double membrane and have a .ell defined nucleolus.

Mitochondria of different shapes ;uith plate-like cristae are

abundant. Endoplasmic reticulum of any ;ind is scarce. Ribosome;

are free in the general cytoplasm. The plasm.aler-ma is elaborated

in plasmalemr.asomes at tar'ious places (Fig. 16). In sore sections

the cells were found full of gltcogcn-l ie bodies (Fig. 13) whilee

in others big globular structure; iere present. inside the sub-

hymenial cells (Fig. 16). The chemical nature of both could not

be ascertained but they appear to be some rind of reserve food

material. The big globular structures are often interconnected and

are full of small light-and dark-c)lored globular bodies. These

also have memb'anous structures inside that run from one to another

globular structure through the connection (Fig. 16).

Closely paced phialides make up the hyrenium. Halfvwa from

the base of the phialide there is a conidiogenous locus where the

wall Is very thick and the conidia are cut off apically into a

collarette (details in Chapter II). The conidiogenous loci of

all the phialides lie at the same distance front the base creating

a distinct thick walled area in the hjneniun (Fig. 7). The

phialides are uninucleate, have riecsores, mitochondria, and rough

endoplasmic reticulum. The nuclei and mitochondria both are very

elongated. Normally four conidia Ean be seen inside a mature

collarette (Fig. 19). The conidia are 3.5-4.5 1.5-2 w, cQ llndrical,

hyaline, thin walled and uninucleale (Figs. 19, 20). The tip of

the phialide is very thick and does not appear to have a pore as

has been reported earlier (Tha.ter, 1920). Discharge of the conidia

was not observed nor was any sporulating sporocarp collected. How-

ever, when a termite with nature famgus was brought into the

laboratory the fungus was found cowved with a white mass of

spores the r.e.t day (Fig. 4). It e.pears as if the whole tip is

blown off the phialide by the force applied by the formation of

new conidia at the conidiogenous lovs.

The cells of the e.cipulum are very thick walled, the outer

surface as thick as 2 L. Though the e.cipular cells in the

hymenium are elongated like conidisquores and are separated from the

underlying cells by typical septa as conidiophores are, they are

sterile (Fig. 22). The cells of the e.,cipulum are uninucleate,

have long mitochondria with plate-like cristae, ribosomres free in

the general cytoplasm, cisternae of endoplasmic reticulum and are

bounded by a tnick wall that is comprised of two layers, the outer

dark and the inner light colored (FiS. 21).

The sporocarp i: traersel by projecting bristles of the host.

The fungus forms a protective filamewous sheath around it (Fig. 10).

The mature sporocarp is a composite structure. The hyphae

appear to emerge at different places and then grow towards each

other (Fig. 61. I think tnat these places dark tne position of

original primsry infe:tion. As far as this study is concerned

these have alr..ys been found lying in a furrow or the inter-

segmental aria. (Figs. 9, 141 where no s leratization occur- and

the cuticle is ver) thin. A11 cells in the basal layer here are

haustorial mother cells sending man) hausroria all around.

Because of its deep lying nature Fe/taud ard Dieuzeide (1927)

called it "pce-icule de l'hi.eriuiT" and Coltl (1929) thought of

it as a foot :o anchor the fungus. Reichensperger (1923) al:o

noted that th: fungus was anchoreJd a..h knob-like intrusions in

the chitinoua matri,.


Because .f the negative response of =-i"'.:1f,_1 to the

media tried for its culture I had to rely crnpletely on field

collections fcr the study of its mode of infection, growth, and


It is assumed that the conidia, like the spores of .some other

entorogernous fungi, become thick walled and high, pigmented

before germination. The cuticle of the termite proves to be 3

barrier to infection. Only the conidia that have come to lie in

the thin intersegmental area appear to initiate the infection.

Not all of the conidia c n find their way to that spot and this

perhaps is why such a small percentage of termites are found

infected. Peichensperger (192I) has suggested earlier that the

infection could occur at the time of molting when the chitir is

still soft. This is still a possibiiit, an. in certain cases it

may be that the infection does start at that time. But the origin

of primary infection in the intersegmental furrow strongly supports

my assumption. The integument does not seem to be trolen at the

time of infection, suggesting that penetration is accomplished by

some enzyre action. It is already known that certain bacteria and

fungi have an enzyme system that can digest insect cuticle

(Richards, 1951).

First, a group of thick walled cells is formed (Figs. 23, 24)

and all of them send haustoria into the host. The hyphae originate

from this mass of cells and diverge in all directions, in

tight lateral contact. The cell at the tip of the hypha is cut off

from the rest by an anticlinal wall, and then divides in only one

plane, periclinal (Fig. 26), thereby resulting in circumferential

as well as radial growth. Thus, the margin of the growing thallus

remains only one cell thick (Fig. 25). During the radial growth

certain cells next to the cuticle of the host modify and become

thick walled haustorial mother cells. It is not clear why one

cell turns into a haustorial mother cell while the next one does

not. There may be certain weak spots in the cuticle and only the

cells opposite them become modified; or perhaps after reaching a

certain size the fungus has to send haustoria; or it may be that

the growth of the fungus is cyclic like that of certain fungi in

agar culture and haustorial mother cells are formed at the beginning

of each new period of growth. Termites, like other arthropods,

have minute ducts extending vertically through the procuticle. These

are called pore canals and one might speculate that haustorial

passage cccurs through them. By observing Fig. 13, a pore

canal and the hiustorial path through the cuticle lie in the same

vicinity, it can be seen that the ha'jstjrial passage appears to be

at the original site of a pore car,n. Thus, it is possible that

only those cells that are opposite pore canals modify, into haustorlal

mother cells. But the dianrater of the pore canals is very small

compared to that of the haustorial path and it array be suggested

that after the initial penetration certain enzymatic activity widers

the canal.

The young thallus is represented by Figs. 14 and 7. It

forms a circular or variously elongated cruit on the host surface.

It is parenchymatous and a fer cells thick (Fig. 25) depending

upon its age. The margin is spreading, only one cell thick, and

remains that way even after the thallus has matured into a mature

spoiocarp (Figs. 17, 26).

The intermediate stages showing the transformation of this

crust-like primary thallus into a sporocarp have not been obtained,

but it appears likely that after reaching a certain size and

thickness the hyphal branches start to grob, upward, very closely

applied to each other. The tips of these hyphae become thicL

called. These are now the conidiopheres, which ;tart cutting

off phialcconidia apically in collarettes as discussed elsehere

(Chapter II) in detail. The peripheral cells teccire thick walled.

remain sterile and male the eocipulum. Thus, the whole thallus

turns into a Soorocarp.

?:.c-it-Co spp. were considered eo.ternjl parasites by Thaxter

(1920), Reichensperger (1923), Colla (1929) and an internal

parasite by Feytaud an. Dieuzelde (1927) and Heim (1952). Theater

did not see any actual penetration but s;a hypertrophied cells of

the host opposite the dark cells. By looking at his figures it is

apparent that he was actually] looking at the haustoria and he

mistook them for hypertrophied host cells. Reichensperger

actually reported that root-like threads emanate from the basal

layer into the epithelium and either perforate the cells or

encircle them. However, he did not realize the importance of

the dar. cells and did not regard the penetration pegs as haustoria.

It is reported here for the first time that the dark cells are

haustorial mother cells and that the fungus is a parasite and

probably is nourished by the host. The sections of the sporocarps

that were observed by light or electron microscope did not show

the presence of any hyphae in the host, only the haustoria that

were uninucleate and did not have any sept, unlike the sporocarp

above the cuticle. N:o haustoria were seen in underlying adipose

tissue or anywhere beyond the basement membrane. The channels

through which the haustoria go into the integument are ,ery smooth

and do not look as if they have been formed by eruption of the

fungus emerging to sporulate, which should have been the case if

the fungus was an internal p.rasite. RTer i-iria2 is an

external fungus that penetrates into the host by haustoria.

The termites with Te;:-i't 'a infection do not look nor behave

abnormally. All the knowledge at hard suggests that the infection

is not lethal. But we also know that the infection of antennae

and legs results in malformation and deformation. PerhapF at a

certain stage of the life cycle the termites are susceptible and

maybe the fungus is lethal. The parasitism of Termitai:..; .-.-1

has been established. The role of this fungus in biological

control for termites awaits more extensive field and laboratory

research. Rearing of termites in the laboratory; ic nece:ar-r to

study the stage it which the termites are moSt suLceptible, howi

much tine it tales for the fungu' to sporulate rrom the tim.- of

the germination of the conidia, whether the fungus lills the

termite; or not, an,3 if it does how it does So.

The geographical distribution is another interesting 3sicct

of the Trnirar:, life cycle. It does not appear to be influenced

by climatic or en ironrental conditions of the area, but corresponds

to the geographical distribution of the hosC anO is such that

almost all the host general ire infected.

The absence of, or our inability to discover, a sejul phase

in the life cycle still leaves the ta.,onomic position of this

unique fungus in dout..

Figures 1-3.

Figure 4.

Figure 5.

Figure 6.


Termites showing the habitat of ? .~'~t.,'L a s:'.der'.
1. Tern7tri'a s:..:lrwi growing on the thorax of the
termite (arrow) .'15. 2. growing on the antenna of
the host (arrow ) .10. 3. growing on the abdomen
of the host (arrow) XIO.

Sporulating X'rrtizria X20. Arrow points to the
white mass of conidia covering the surface of
the whole sporodochium.

Whole mount of terr.ite legs X20. Compare the
infected leg with the uninfected one and notice
the swelling and deformation of the former.

A close-up of the basal layer of the mature
sporocarp '100. Arrows indicate the multiple
points of origin of infection, Raking the thallus
a composite structure.



Figure 7. Cross section of mature sporocarp of ".'r-:,..-i?
ar.'.:dqr showing basal la;er (BL), subhjmenium
(SH), hjTmenium (Hi), e.:ipulum (E), and the
bristle 1() of the termite cuticle passing
through the sporocarp 71,000.

Figure 8. Plastic section of the sporocarp shoiiing the
subhy~.eniui (CH) outside and the haustoria (H)
with distinct nuclei (N) inside the host

Figure 9. Plastic section of the sporocarp at the point of
the origin of infection showing many thick-walled
haustorial mother cells (H1C I and nucleate (N)
haustoria (H) .2,000. One of the haustorial
mother cells is connected with the underlying
haustorium (arrow).

Figure 10. Cross section of 3 portion of sporocarp X2,000.
Bristle (B) of the termite cuticle passes through
the subhmenium (SH), hymenium (Hi) and is surrounded
by the sterile filament (arrow points to one such

-me i.J~


Figure 11. Haustorial r.:.
peg into the
hjustorial r:.
(ER), mitocc
separated r ,..
a typical as .
(W) in the vi:

Figure 12. Light micro.,r
distinct h.,3

ell (HMC) sending a penetration
.e cuticle (CLI) \22,500. The
1ll has endoplasmic reticulum
(11), a nucleus (N) and is
cells of the subhymenium by
.e septum with Woronin bodies

- haustorial mother cells with
-.ots in their centers x2,0013,.






Figure 13. A near median section through the neck (n) that
connects the haustorial mother cell (HilC) with
the haustorium .(14,100. The neck appears to
pass through a pore canal, since its passage
through the cuticle (CU) looks similar to the
pore canal (PC). Haustroium has a thick -'all
(W) and contains a nucleus (ri) with a single
nucleolus (NU), mitochondria (i), and endo-
plasnic reticulu (EP.).

Figure 14. Young thallus of Tcr-r-i.:.a growing on an
antennal of the termite Y200. It
appears to originate at the base of the
segment ani upward in many directions.
Dark haustorial mother cells (HM.C) are very

Figure 15. Cross section of the antenna segment showing
it surrounded by the fungus crust :?50. The
bristles (B) of the termite cuticle are passing
through the young thallus. Basal layer of the
thallus is very dark and prominent.




.U4 N L


EtvJ'B'~ q y
I -I
0 V

( ^ ) -Bi*0


Figure 16. Subhymenial cells of the sporocarp separated from
each other by a typical ascomycete septum having
a central pore (arrow head) and Woronin bodies (W)
.433,000. Plasmalerra is forming plasmalemasomes
(PL). Subhirienial cells have mitochondria (M1)
with plate-like cristae, ribosomes in general
cytoplasm, and globular structures (GS) that have
membranes in them that sometimes run from one
globular structure to another (arrow).

Figure 17. Saggital section through the margin of mature
sporocarp X2,000. Note a single cell at the

- ,N


Figure 18. Excipular region of tne sporocarp showing
subhymenial layer and the base of the hynenial
excipulumT X15,000. The cells are filled with
glycogen-like bodies (G). Some of the sub-
hmenial cells have vacuoles (V), globular
structures (GS) and one of the cells has a
nucleus (N). One subhvnenial cell is connected
to two sterile hyrenial cells of the excipulum
with ascomycete septa having central pores
(arrows) and Woronin bodies (1).

B ~

:' N 18
~' 2'



Figure 19. This section of the hyjr.enum showing conidiophores
(CP) and the excipulum (E) X3,000. The former cut
off conidia (C) with nuclei (N) into the collarettes
(CO) at the conidiogenous loci (CL).

Figure 20. Whole mount of conidia X1,00.


E~-IEI~~ C~I~ ('~~4


Figure 21. Thin section of the e. sporocarp 037,500. The cell is thick walled,
has a nucleus (N) with a nucleolus (NU) and
mitochondrion (N).

Figure 22. Cross section of the e.'cipul3r region of
sporocarp showing thick-walled cells in the
ecipulurn (F) and a portion of the h.renium
(HY) X2,000.


' -. ^-'.)--

if .-


Figure 23. A section of the very young thallus comprised of
only a few thick-walled cells growing on the host
cuticle (CU) X2,000.

Figure 24. Whole mount of a very young thallus comprised of
thick-walled cells that has just started sending
hyphae around itself X2,000.

Figure 25. Cro'-s section of a young thallus a few cells
thick in height growing on the host cuticle (CU)
and sending haustoria (H) into the integument from
its haustorial mother cells (HIIC) Y2,000.
Spreading margin is only one cell thick (arrow).

Figure 26. Surface view of the growing margin of the young
thallus X2,000.

Figure 27. Whole mount of a young Ter.t .:a thallus viewed
from the top 2~50. Neatly arranged filaments
originate from a group of thick-walled cells
(arrow) in all directions. Dark haustorlal
mother cells (HMC) are arranged in rows.

- fI-, -,

*i.Au 4




Hughes (1953) emphaSized that "morphologically related imperfect

states can be brought together wrien the precise methods of conidium

origin take first place in the delimitation of the major groupings."

Since then considerable attention has been focused on the structure

of conidiophores and the mode of conidial formation (Tubaki, 1952

and 1963; Sutramanian, 1962). Hughes classified the Hypomycetes

into eight large sections, each based upon different mechanisms of

conidiogenesis. In section IV of his classification he included

those Hypom)retes that have conidiaa developing in rapidly

maturing basipetal series from the apex of a conidiophore which

may or may not possess an evident collarette." ,'T.-.ri

belongs in this section. Hughes restricted the term "phialide"

to those unicellular structures which are usually terminal but

sometimes intercalary as well, on simple or branched conidiophores.

They are oval to subcylindrical to flask shaped or subdulate, often

with a well differentiated basal swelling and a narrower distal neck,

with or without a terminal collarette. From the ape* of each

phialide develops a basipetal succession of phialaspores without

an increase in the length of the phialide itself. This section

corresponds to the Phialasporae of Tubaki (1963) and Tuberculariaceae

of Subramanian (1962).

Cole and Fendrick (1l69) studied the phial ides of i'-ialaphora

;,j-r;r,'j:: (f'lel in and IJannf.) Conant, F- i.e: :. .:'d ophilZtm

Dierckx and Th:'..:.::.:p-r.; ,:r,:::. (De Seynes) vro Hohrel by time

lapse photomicrography. In all three they found a fixed endogenous

meristei responsible for conidium formation. The conidiophore

ceases elongiaion once the Teri:tiim become: active and its outer

wall is ruptured at the ape\ by the emerging first conidium or its

initial. In each case a portion of the conijiophore wall rerrains

above the meristeematic one and acts as a collarette through which

the conidiu.i or its initial protrudes. They defined the phialide

as "a sporogenous cell with only one functioning, fixed, endogernous

meristem whose position is marked by the deposition of an inner cr

secondary wall which surrounds each of a basipetal succession of

physiologically independent conidia. Spore production incurs no

concomitant increase or decrease in the length of the iporogenous

cell." At the recent Kananaskis conference on Ta.,onomy of Fungi

Imperfecti (Kendrick, 1971) the phialide was defined as 'a

conidiogenous cell in which at least the first condium initial is

produced within an apical e.,tension of the cell but is liberated

sooner or later by the rupture or dissolution of the upper wall of

the parent cell. Thereafter, from a fixed conidiogenous locus a

basipetal succession of enteroblastic conid a is produced, each

clad in a newly deposited wall :o which the wall of the conidiogenous

cell does not contribute. An, phialide wall distal to the conidiogenous

locus is the collarette. The length of the phialide does not change

during the production of succession of conidia."

Materials and Methods

The subterranean termites Ef:c:icitceez spp. bearing typical

Termitzr.2 lesions were collected from the woods around GainesJille,

Florida. The lesions were removed along with the adjoining

integument, under 2.5% buffered (I:a-Cacodylate pH 7.35) gluteraldehyde

and fixed for four hours at 3VC. The material was washed in buffer

and post-fixed in buffered 1.5. osnium tetro.ide overnight in the

refrigerator. After several rinses in water it was dehydrated in

a graded series of ethanol and in the end washed in reagent grade

acetone. It was infiltrated with graded acetone-plastic mixtures

and finally embedded in 100l plastic. Mollenhauer's (1964) plastic

mixture #2 was used (62 ml Epon 812, 81 ml Araldite 506, 3-4 ml

Dibutyl phthalate; 15 ml of this mixture, 10 ml DDSA and 45 drops

of DIP 30 to make the final plastic mi.ture). For better penetration

the material in the miAture was put on a shaker for eight hours, at

every change during the graded acetone-plastic mixture series.

Bubbes in the plastic were removed in vacuum at 60C. Polymerization

of the plastic was carried out in an oven at 60C for three days.

The material was stained for two hours in 2% UAc in 70; ethanol

during the dehydration and post-stained in 0.5% aqueous UAc for

45 minutes arw with lead citrate for 30 minutes. Some grids

were stained with methanolic UPc as described by Stempak and Ward


The sections were cut on a Porter-Blum MT-2 ultramicrotome

with a diamond knife and examined with a Hitachi-HU 11 E electron

microscope. Some half-micron sections were cut and stained with

NIl (Juniper r ,J:., 1970) for light microscopy. Cryostat sections

of the sporocarp were cut without fixation and stained with aniline

blue in Hoyers. Some whole lesions were mounted in Hoyers and

aniline blue, crushed, and ob-erved under the light microscope.

The conidia are cylindrical 3.5 4.5 u 1 1.5 2 u (Figs.

30, 31, 32, 41). The conidial wall is composed of two distinct

layers, in outer thin electron dense layer and an inner thick electron

transparent layer (Figs. 31, 41). Like other fungal spores the

conidium surface is smooth. Conidia are uninucleate, the nucleus

is bound by a double-imeirtrane, nuclear envelope. Usual or;anclle:

sucn as mitochondria, rough endoplasmic reticulum and ribosoTrie; are

present (Figs. 31, 43, 43). Many lipid droplet; are also present.

In certain conidia the droplets look interconnected (Fig. 43).

Unusual vacuoles have been seen in some conidia (Fig. 31).

The conidiophores are long, cylindrical, vertically parallel

in a sporodochium (Fig. 29). They appear to te fused laterally

with each other, giving a honeycomb appearance when viewed from the

top. The conidiophores are 55 65 p long and 2.0 2.5 u wide,

with a very thick ]nd rounded tip (.ig. 40). Less than half way

down from the tip is the conidiogern s locus (Fig. 28). The part

of the conidiophore is constricted at the locus forming a very

short neck (Figs. 34, 37, 43). The conidiophore wall in the region

of the collarette consists of two layers, an outer more electron

dense, and inner more transparent layer. We could not differentiate

the layers in the rest of the conidiophore wall. At the base of the

collarette there is a pad of wall material between the outer and

inner layers of the wall (Fig. 47). The apparent thickening in the

neck is actually the inner wall la)er of the conidiophore that has

been sectioned obliquely as is evident by the similarity in electron

density. The plasmalemma of the conidiophore is highly convoluted

forming praramral bodies (ilrchant and Robards, 196S) all along the

length of the conidiophore. Sometimes connections can be seen

between the vesicle of paramural body and the plasrralemma (Fig. 35).

The coniiophore is separated from the vegetative cell under-

neath by a septum perforated by a septal pore. There are darkly

staining Woronin bodies near the septum (Fig. 42). The Woronin

bodies are bewnded by a unit-membrane and have a granular matrix.

Plasmalenmasomes h3ve been seen projecting into the protoplast of

the cell underneath the conidiophore (Fig. 39).

Each conidiophore is uninucleate, has mitochondria, endoplasmic

reticulum, ribosomes and vesicles (Figs. 33, 34). The nuclei and

mitochondria are very elongated. Each nucleus is delimited by a

double membrane and has a conspicuous nucleolus at one end. The

nuclei of all the conidiophores are situated halfway from the

conidiogenous locus, and we found all of them in interphase stage

even though in many cases conidium initials were forming at the

loci (Fig. 28).

The conidium initial buds out at the locus into the collarette.

The wall of the conidium initial appears to originate within the

neck of the crnidiophore (Fig. 47). After the conidium initial

has reached a certain size and cell organelles have moved into

it, a delimiting septum starts growing centripetally near the

conidiogenou: locus. The septum has a central electron-transparent

layer between two electron dense ones (Fig. 47). At the rim, of

the growing septum an electron-dence material is present (Figs. 45,

46). The septumT appear: to become functionally complete by

continued cein.ripecal growth (Figs. 44-43). Most of the septum

grows at the conidiogenous locus except for a central pore that

remains open untill the ne.w conidiun directly underneath is fully

grown (Figs. 36, 38). The thickness: of the conidium wall does not

increase noticeably during its forration. However, after it has

become delimited by the septum an inner electron-light wall layer

is deposited Shat grows in thickness as the conidium matures (Fig.

41). The young conidium is connected with the newi initial through

a central pore in the septum (Figs. 36, 33, 41). After the septum

is complete the electron-light layer of the septum appears to

break down, allowing the two halves of the septum to separate and

the conidiuim to secede (Figs. 47, 48). In rare cases Ve have seen

Woronin bodies near the growing septum (Figs. 36, 46, 47). The

shape of the young conidium is determined by the tubular collarette

and the constricted conidiogenous locus. The young conidium is

typically narrow at both ends (Fig. 43) because the septum at

both ends originates at the constricted conidiogenous locus.

From the above observations we reach the following conclusions:

1. The conidia are produced in basipetal succession from a

fixed coniditgenous locus.

2. The conidia are clad in an entirely new wall not derived

from any existing layers of the conidiophore wall.

3. There is no increase in the length of the active cor.diophore

i.e. from locus to the base of the conidiophore.

Conidium Initiation

I am aware of only three reports of the type of Phialoconidio-

genesis where the conidia are produced in a tubular collarette. Tuo

of them are of species of T;:hciaiopfi. Cole and Yendrick (1969)

have studied phialoconidium ontogeny of ". p:,.ariz.?2 (De Seynes)

von Hohnel by time lapse photomicrography, and Del Vecchio .a .Z.

(1969) of r. bsicola by light and electron microscopy. The

conidiogenesis of an Indian isolate of 7. paradc was studied by

Seshadri as reported by Subramanian (1971).

According to Cole and Yendrick (1969) the phialide of T. a'~. :'

is long and cylindrical. One to several conidia differentiate

basipetally within the conidiogenous cell. During early stages of

differentiation growth of the outer wall of the conidiogenous

cell and the conversion of the protoplast into conidia proceeds

simuTtaneously. The center of activity (conidiogenous locus)

continues to move downward until the conidiogenous locus is fixed.

The phialide stops growing and the continued production of the

conidia at che fixed locus e.erts a pressure at the tip of

phialide resulting in the rupture of the vall thereby releasing

the conidia. The cylindrical portion of the phialide beyond the

locus is a collarette. The locus is not marked by any constriction

or other morphological evidence of the phialide wall. The

conidiophore does not elongate after its apex has ruptured.

Seshadri's (S4iAramanian, 1971) work confirmed Cole and Kendrick's

(1969) findings. Moreover, Subramanian (1971i reported the conidia

did not appear to be delimited by a process of septation or double

septation in the Indian isolate of T. paradoxa.

Del Vecchio et at. (1969) described phialides of T. basicola

enclosed inside "hyphal tubes." Tney contained endoconidia and

were composed of cells which were always devoid of cytoplasmic

components. The walls of these cells were never attache. to thE

walls of the endoconidia an, were identical to the cell walls of

the vegetative hyphae. We think that they risinterpreted the

tubular collarette as a hyphal tube. It becomes clear by looking

at their Fig. #1 Plate 1 that the conidiogenous locus is deep seated,

is not marked by any constriction of the phialide -.all, and the

conidia are released by the rupture of the phialide tip.

Subramanian (1971), by looking at tte reports of Cole and

Kendrick (1969), Del Vecchio t a:. (1969), and an Indian isolate

of the r. pmr.~I:& z, suggested that the events of conidiogenesi: are

as follows. The first step in the development of each conidium

appears to be the protoplasmic cleavage at the conidiogenous locus

within the phialide, followed by development of a totally new wall

around the cleaved mass. This process goes on in production of

several conidia in basipetal succession.

Our findings agree with those of the above in the presence of

tubular collarettes, the wall of the conidia being separate irom

that of collarettes, the phialide wall not contributing to the wall

af tne conidia, there being no increase in the length of the phialide

after its apex has ruptured, and that the conidia are produce- in

hasipetal succession.

As opposed to the findings of others, the conidiogenous locus

in the remnnirza ,i:eri phialide is marked by a constriction in its

wall. It appear; as if after reaching a certain stage the conidium

initial buds out through the thick walled tip of the phialide but

the phialide wall is not broken (Fig. 49 a and b). Thus, a

conidiogenous locus is established, marked by a constriction. The

conidia are produced in basipetal succession from this locus (Figs.

41, 49 c-e). The wall of the conidium appeared to originate at

the locus as has also been reported for f,'l.vrpor.i cracc by Lowry

et a1. (1967), for 'ercic: :iin a io-2:rwn by Buckley et .;:. (1969),

for PF-ni:.lin c-jitfci"C by Zachariah and Fitz-Janes (1967) and

for tit.zf'lhiZcn :;deplia. by Harmill (1972a). The growth of the

wall of the phialide and the formation of conidia proceed simultaneously

during early stages. However, we presume that after a while the

phialide stops growing and the pressure thus exerted at its tip

results in the rupture, releasing the conidia. After that, no

increase in the length of the phialide takes place. We were unable

to see the formation of the first conidium initial as well as

rupture of the phialide, because the fungus does not grow in

culture and we could not collect it in those two stages of develo-

ment. However, Thaxter (1920) has reported that the conidia are

released by the rupture of the phialide tip.

The growth of phialides and formation of conidia looks quite

synchronized as is apparent in Fig. 28, where the conidiogenous

loci of all the phialides appear to be formed at more or less the

same level, resulting in a clear cut zone in the sporodochium. All

the phialide: are of the same helgL.r and mo:t of them have four

conidii. This type of synchronous deseloprTent of adjacent phialides

has also been reported by Trinci1 :: c. (19.S) for 4zi :;.- .;z:i...

The rmde of conidial deli.-.itation is also different from that

of ". par'.:= 1a and T. -az:-'.--:;. It is by double septation. The

septum grows centripetally and for quite a while has a central pore

which may or ray not have Wloronin bodies in there vicinity The

septal pore closes by centripetal growth. We have not seen Woronin

plug: in ? ri.:. f lire those reported by Cole and Aldrich (1971)

for 4:;: K.n.ic;r.' te.."',c':f: (Sac:.) Bain. However, the

septun is similar to the septum reported for b. .i;.:..-i.:.'z by

Cole and Aldrich in thit it has an electron transparent layer

sandwiched between two electron dense layers (Fig. 47). Hanmrill

(1972b) reported a similar type of septum in 0 ::.:-'.m-~c-. ,-:. but

as the conidiogenesis continued the electron transparent layer

underwent a transition and became mere electron dense than the other

two. Buckley er *a. (1969, Fig. 12) also show the same construction

of the basal conidiuTm septumT of i'-ri-d:- L:in atc.-.:r., an electron

transparent layer between tio electron dense ones.

The dividing septum undergoes lysis along its aris as has also

been reported for budding of yeast rF--.;::lr;.c: 2:;.':'z by Marchant

-t al. (1967a.Fig. 21).

Thus, phialoconidiogene-is of .'- rn:'r-.2 z:J,-;'. Thaster differs

from that of 7;:'ic.',- .:s p.-tccr~ an.d ",Z;:oZ.U in the following

very important details: (1) the conidiogenous locus is fi>ed in the

beginning; (2) it is marked by a constriction in the phiallde; and

(3) the conidia are delimited by a double septum.

Fletcher (1971) reported that the conidial chains of three

different species of FEr.cilic: F. aiiger:,.. Damelius, P. c c:, w.?

Bainier and P. cn.Fr~crji n Westling, were enclosed within an

electron opaque surface layer that appeared continuous with the

surface layer of the phialide wall after gluteraldehyde and Osj4

fixation. He did not say whether he considered it as a collarette

or not.

Growth of Conidiophores

Several schemes have bee:i proposed to explain the phenomenon of

apical growth. According to Eracker (1967), Grove et a_. (1970a and

1970b), fkClure e: a'. (1968), cytoplasmic vesicles are present to

the exclusion of other organelles in the very tip of the hyphae.

Grove et al. (1970b) reported that the single cisternae Golgi play

an important part in the vesicle formation. The vesicles are forced

posteriorly, migrate to the apex, fuse with the plasma membrane, and

liberate their contents as part of the process of growth. However,

Marchant e: al. (1967a) found two different vesicular systems

associated with w3ll synthesis in regions of active growth and

wall synthesis. In the apical region of the hyphae vesicles pro-

duced by the endoplasmic reticulum moved to the plasma membrane,

fused with it, and were responsible for the primary wall formation.

In the older region of the hyphae multivesicular bodies were found

that fused with the plasma membrane and gave rise to lomasomes and

were apparently associated with secondary wall syntnesis. It was

questiornci whether all the loadsoi;e-1l e bodies were honologoI: in

structure and function. Therefore, Marchar.t -;. ,:. (19 ?) proposed

later that all the memibrarous or vesicular structures associated with

the plasma nerbrane be called paraitral bodies, that the uord

loadsore be limited to the structures deri ed from cytoplasnic

vehicles or mulctiesicular bodies, and that all structures derived

from the plasMaleama be called plas..aleiT-rasom.-. They also suggested

that lo,.isomes might be involved in the incorporation of wall pre-

cursors by transport across the plasmale.iia in th-e same ,ay as the

single vesicles originating from ER and Golgi do.

The parai.-ral bodies have also been regarded as artifacts of

fixation and their existence in live iiaterial and their role in

wall synthesis has beer questioned. Recently, evidence that the

paraTural bodies may not be artifacts comes from wor.k of different

people. Griffiths (1970) found theA in frozen etched replicas of

hyphae of rve:cici::s.! ,.:i. Kleb. Heath ,t S:. (1970) demonstrated

their presence by using three types of fiations in the wall of the

growing hyphae of :pr.-::ic fe (ra.-r (Gruithuisen) Thuret and in the

walls of primary spores and their exit papillae in Di.accyic-.'

:-.ri>z Coker. They also used SITS stain which is claimed to bind

to specific sites in plasmalem i After incubation in SITS stain

a strong pale blue flloresence was detected in all hyphae, most

intense at their apices, sometimes occurring in diffuse patches,

the patches rmarking the sites of planmalem-maomes. They also

suggested that the plasrralemrasomes are produced r.hen more plasmalerima

is produced than is needed to line tre cell wall and the plas-,alemmasomes

may become sequestered in tne jevelcsing wall.

The paramural bodies have been reported in iLrrti-;;::;.X a:;.-at;:,.

at the double septum by EucFley 6t a:. (1969), and in 4srEer'":l:,

n:., i ans in the subapical and basal cytoplasn of growing sterigmata

and near their developing septa by Oliver (1972). Multivesicular

bodies have been seen in .'"e:-rr;:=tz:. ,c:' by Harinill (1972a)

at both sides of growing delimiting septa whEn wall material was

being deposited, which suggests that they may have played a role in

its deposition.

The cytoplasmic vesicles have also been reported associated with

conidiogenesis. Trinci t aL. (1968) reported the presence of

vesicles in some sections inside phialide primordia. At the time

of bud formation in Rhodc .r.lj gl:,:;'.-, Marchant ae a (196bi)

reported vesicles in the bud at the time of active Y.all synthesis.

Carroll (1972) found fibrillar material and masses of gray and

speckled substances associated with plasmalemiasomes at particular

stages of conidiophore and conidiun development and near growing

septa of te Ip..h2,:L: kL.rlryca '. She supported the conclusions of

Marchant ot :;. (1968) that plasmsilermasomes nay be involved in

secondary transformation of wall materials.

In Territrr-. An.e!" ThaYter phialides the paramural bodies

are present all along the plasmalenma. However, here the tip of

the growing conidium initial is filled with the vesicles (Fig. 10,

16, 18, 19). The vesicles are seen only in the vicinity of tne

growing delimiting septum and only at that stage. After the septum

is compelte in the old conidium initial they are not seer.. Their

location and timing suggest that they may be involved in the synthesis

of the septum.


I agree with Heath and Cre.neood (1970) that piasmalemmasomes

are produced when more plasmalemma is formed than is needed to line

the cell wall. I suggest that at :t.e time of conidium production

the convoluted plasmalemma stretches a.nd covers the initial and

helps in the formation of it: first wall. I have not seen any

fibrillar or gray or specked -ub:tances associated with the

plasma emma some.


Figure 23. Cross section of a portion of the mature sporocarp
stained with AMB (2,000. It shows the haustoria (H)
beneath the cuticle (CU) and the subhymenium and
hyrrenium above it. The basal layer has many thick
walled haustorial mother cells (HMC). The conidio-
phorez (CP) have elongated nuclei (II) and prominent
conidiogenous loci (CL) all of which lie at the same
distance from the base of the hyenium making a very
distinct line across the hymenium.

Figure 29. A mature sporocarp on the dorsal side of the abdomen
of a termite (arrow) (20.

Figure 30. Whole mcunt of conidia under phase contrast Y2,000.
Lipid droplets are distinctly visible.

Figure 31. Thin section of a conidium inside the collarette
showing outer (OL) and inner (IL) layers of
conidial wall and lipid droplets (L), vacuole
(V) and nucleus inside the conidium X28,000.

Figure 32. Whole mount of conidia under bright field Q2,000.


.-. ..,-+ -. I-- L ,"
Yi'li -

frb a1


Figure 33. Thin section through the conidiophores /15,000.
Elongated nuclei (0) with nuclear envelope (NE)
and nucleolus (;U) are present in the conidiophores.
Surface view of the plas.malerma of two conodiophores
shows plasmalemmasises (PL).

Figure 34. Thin section through the apical region of the
conidiophores X15,0GO. Conidiogenous loci (CL),
elongated ait3chondria (M) and rough endoplasmic
reticulum (ER) are evident.

Figure 35. Plasma membrane (PM) of the conidiophore with
plasmalemmasoTes (PL) X88,000. Arrous point to
the connections between plasma membrane and the
plasmalemmasomes. Cisterna of rough endoplasmic
reticulum (ER) is lying close by.

., .: .. 1

-'" ,. t


r L.


Jr. ^ *


Figure 36. Thin median section through the conidiogenous loci
showing budding and growing conidia 715,000. Arro,
points to the connection between the budding and
maturing conidiun. lMitochondria (M), lipids (L)
and a Woronin bod. (W) are also there.

Figure 37. Thin section through the tip of the conidiophore
filled with vesicles (VS) X62,000.

Figure 38. Thin section of young and maturing conidia inside
the collarette X40,000. Arrow points to the
connection between the two conidia. ;ote that
the conidial wall is separate from the w~ll of
the collarette. Lipids (L) are present inside
the conidia.

Figure 39. Thin section through the septum between the conidio-
phore and the cell underneath X52,000. A big
plasmalemrasone (PL) connected with the plasma
membrane is projecting into the cell cytoplasm
and a Woronin body is present.

~1~ j


.a~ i~j

lope%, S-'


Figure 40. A near median section through the tip of the
colljrette .19,200.

Figure 41. A near median section through the conidiogenous
locus (CL) and the collarette (CO) having conidia
in different stages of maturity Y3,500. The
conidla are uninucleate (N).

Figure 42. A thin section of the septum at the conidiophore
base .'.94,000. Arrow points to the unit membrane
around the ol'ronin body (W). Plasma membrane (PM)
is continuous around the edge of the septum towards
the pore.

Figure 43. Thin section through a youngest conidium of the
chain ,'24,500. The conidium has a nucleus (I0)
bounded by a nuclear envelope (NiE), rough endo-
plasmic reticulum (ER) mitochondria (M) and
lipid droplets (L) that are interconnected. Apex
of the conidiophore is filled with vesicles (VS).
A Woronin body (W) inside the conidium is also
present. Note that the young conidium tapers
toward both ends.


> 1i!

*'-* '


7 %.


Figures 44-48.

Approximately median sections of the conidio-
phores and the collarettes representing
different stages in the formation of double
septum and secession of conidia.
44. Young septum originating at the conidio-
genous locus \28,000. Notice a mitochon.jrion (1)
between conidium and the conidiophore. Lipid
droplets (L) and rough endoplasmic reticulum are
present in the developing conidium. 45. Later
stage in septal growth X30,000. The tip of the
conidiophore is filled with vesicles (VS). Arrow
points to the electron dense material. 46. Slightly
later stage than the previous one in the centri-
petal growth of the septum X52,S00. Arrows point
to the electron dense material at the rim of the
growing septum. Vesicles (VS), Woronin bodies (l)
and lipid droplets (L) are also there. 47. Fully
developed double septum with a central pore Y40,000.
Black arrow points to the electron transparent layer
of the septum while the white one indicates the
point of conidium wall formation. A granular
material is present between the inner (il) and
outer (ol) layers of collarette wall. Lipid
droplets (L), Woronin bodies (W), and rough endo-
plasmic reticulum (ER) are also there. 48. Conidial
secession X48,000. The arrow points to the place
of secession-the electron transparent layer of the
double septum.





- -I


Figure 49. Diagrammatic interpretation oi seq'uerce of conidio-
genesis. a. conidiophore tip. b. budding of first
conidium and forr.ation of conidiogenous locus tCL)
and the collarette (CO). c. sop:am initiation at
the base of the conidiuri. budJing of second
conidium, first septum is complete e-ccpt for a
central pore. e. first conidiuri is seceded irom
the one underneath, a new.. inrer layer (IL) is
being deposited inside th". outer one (OL) of the
conidium will, a third conidiuja is starting to
bud out at the conidiogencus locus (CL).


L~___ -- 1



Studies about fungal haustoria inside the insect hosts are

scarce. Richards' work about Herpnr-gee infecting cockroaches is

on the light microscope level. However in recent years a number of

workers have published about the haustorial structure inside plant

hosts (Hawker, 1964; Bracker, 1967; Ehrlich and Ehrlich, 1971). A

generalized concept of the host parasite interface, with a few

exceptions, has emerged. A haustorial mother cell, in most cases

thick walled, is present. The haustorium and the haustorial mother

cell are connected by a narrow neck. The fungal wall is continuous

around the neck, an exception being Albu:qc ndti~ haustoria which

lack part of the wall around the distal end of the neck (Berlin and

Bowen, 1964). Haustoria are uninucleate in all cases except for

Albqg orid, FP t.:pk 'r,; ir.festwias, P. pararit:ica, P:F;:ersv.cpra

cuberasi (Ehrlich and Ehrlich, 1971). The haustoria are surrounded

by a sheath (Encapsulation or Zone of Apposition) and are separated

from the hcst protoplast by a membrane that is continuous with the

host plasma membrane.

In this study the haustoria of the entomogenous imperfect

fungus Te-rnt.;ria e :.dri Thaxter infecting the subterranean

termites Re-Kec itr-ea spp. were investigated. The fungus forms

lenticular to hysterioid sporocarps on the exoskeleton of the host

irre:picLvi'e of thi body position and is found closely appressed to

the cuticle. Tne basal laer of the ;porocarp is comprised of thin-

wa;lcd cells intersperred uith thicker-balled, bigger, darK-colored

cells. These cells act as haustorial mother cells.

la trials and 'etrods

Tre subterranean termites ,c:,i;.'t: ,rr ..: spp. bearing typical

ri:..7arI' lesions were collected from t e woods around Gainesville,

Florida. The lesions were removed along with the adjoining integuiTent

under 2.51 btiffred (Na-Cacodylate pH 7.35) glutaraldehyde and fi.ned

for I hours zt 4VC. The material was washed in buffer and post-fi.,ed

in buffered T.5, osmium tetroxide overnight in the refrigerator.

Afrtr sc.vera'. rinses in water it was dehydrated in a graded series

of athnrils and in the end washed in reagent grade acetone. It was

ir.fillrated H itch graded acetone-plastic mir.itures and finally, embedded

in 100. plastic. Mollenhauer's (1964) plastic mixture 42 (62 ml

Epon S12, 81 ml Araldite 506, 3-4 ml Dibutyl phthilate; 15 ml of

this mi.ture, 10 ml DDSA and 45 drops of DMP 30 make the final

plastic ni.'ture) was used. For better penetration the material was

put on a shaker for eight hours at every change during the graded

acetone-plastic mixture series. Bubbles in the plastic were reToved

in vacuum at 50"C and the plastic pol)ynerized at 60'C for three days.

The material was stained for two hours in 21 uranyl acetate in

701 ethanol at room temperature during the dehydration and post-

stained irn C.5 aqueous uranyl acetate for 45 minutes and with lead

nitrate for 30 minutes, on the grids. Some grids were stained with

methanolic ursanyl acetate as described by Stempak and Ward (1964).

The sections ..ere cut on a Porter-Blum MT-2 ultramicrotome with

a diamond knife and examined nith a Hitachi-HU 11E electron microscope.

To determine acid phosphatase activity the rcr- t:p.2 lesions

were fixed in glutarzldehdde as described above. After fixation the

material was rinsed in Nra-Cacodylate buffer (7.35 pH) four times,

15 minutes each. Then it was incubated in a modified Gomori's

Medium (Barka and Anderson, 1962) at room temperature for 30

minutes at pH 5. After incubation it was washed in Na-Cacodylate

buffer at pH 7.35. It was later post-fixed in 2% osmium tetro.ide

buffered at 7.35 overnight at 4C. After that it was dehydrated in

a graded ethanol series and embedded in an Epon-Araldite mixture as

described earlier. The sections wre cut, stained and observed in

similar manner as for general haustorial study. Control experiments

included incubation at pH 7.0, incubation without 6-glycerophosphate

and incubation without lead nitrate.

Periodic acid silver stain (PAS) (Martino and Zamboni, 1967)

was used to test for the presence of polysiccharide. Four different

aqueous solutions were made: 2% periodic acid, 3% examine, 5t

silver nitrate, 5' borax. The staining solution was prepared

shortly before use by mining 23 ml of hetamine, 25 ml of AgNrO3 and

4 ml of borax. The solution was centrifuged at 2,000 rpm for

30 minutes. Grids containing sections were transferred to the sur-

face of periodic acid solution for 15 minutes at room temperature.

Later they were rinsed twice nith water and were floated over the

staining solution at 60C for 30 mites. After staining, the

grids were floated individually upon hypo for 30 seconds and

later rinsed in water. orne grid: containing sections were stained

without prior oxidation by periodic acid.

The haustorial mother cell is very thick walled (Fig. 50, 52,

53). The cell wall is distinctly divided into two zones the outer

dark colored and the inner light colored. The inner light-colo'ed

zone may be comprised of many layers. The haustorial mother cell

is anucleete, has mitochondria, lipid bodies, v3cuoles, Cisternae

of smooth and rough ER, ribosomes and microtodies. Ver) rarely some

of the haustorial mother cells are uninucleate (Fig. 50). The

nucleate haustorial mother cells were not found connected to the

haustoria and it is difficult to say whether the haustoria belong-

ing to then were nucleate or not, because serial sectioning could

not be done, since sections of this material tear very easily.

The mother cell is separated the adjacent fungal cell by a

typical ascomycete septum having Woronin bodies in the vicinity.

The mother cell is connected to the haustorium underneath the cuticle

through a neck and its inner wvall ler is continuous around the

neck to the haustroium (Fig. 52, 53). Mitocondria, unique 80 A

microtubules, normal microtubules, and ribosomes were seen in the

neck region. The hole in the cuticle through which the neck runs

is very srooth. Often at the base of the haustroium there 1i a

collar of some granualr material (Fig. 52) in which no membranous

structure could be resolved. The electron density of the collar

is the saze as that of the innermost layer of endocuticle.

The haustorium is a branched structure having its only nucleus

in the base. The nucleus has a nucleolus and is surrounded by a

membrane envelope (Fig. 53, 54, 55). Certain me-bbranous structures

are present in the nucleoplasm.

The wall of the haustoriun is two layered with the outer being

darker than the inner layer and being PAS positive (Fig. 53, 62).

The inner layer of the wall is continuous with the inner la er of

the haustorial mother cell wall.

The haustoria have not been found in direct contact with the

host protoplast but are separated from the host protoplast by a

membrane that can be traced to the host plasmale.1ma.

The haustoria displace the epidermal cells and make room for

themselves (Fig. 54), thereby resulting in the localized swelling

of the body part the fungus is growing on. The fungus evidently

does not penetrate beyond the basement membrane of the host


The older haustoria appear to be hanging in the space they

created for themselves, and are seen in close contact bith the

host protoplast only apically (Fig. 54). However, the younger

haustoria may be surrounded by the host plasma membrane. In older

haustoria the space between the haustorium wall and the membrane

separating it from the host is filled with fibrous material (Fig. 56).

This material is PAS negative (Fig. 62) suggesting that it is not a

polysaccharide. I tried pronase digestion but due to certain

technical problems could not reach a conclusion. However, I

think that it is proteinaceous in nature. In the young haustoria

either there is no such space (Fig. 59) or a very small one. The

host cells around the haustoria haie the usual organelles:

mitochondria, ritocones, eni jla;mic reticulum, nuclei, microtubules.

The most common organelles closest to the haustoria are the micro-


The haustoria have Titcchondria, microtubules, microbodies,

endoplasmic reticulum, lysosomes, vesicles, multivesicular bodies,

and nuclei. The haustorial may form plasmalemmasomes

projecting into the cytoplasm of the haustorium (Fig. 56).

Autophagic vacuoles have been seen with ER, mitochondria

(Fig. 72), and lipid (Fig. 71) inside. The autophagic vacuoles or

lysosomes are surrounded by a unit membrane, and are filled *rith

debris and vesicles.

Both smooth and rough endopl]:m'c reticulum are present.

Smooth endoplasmic rEticului is stacked and its citccrnae are

continuous with those of rough Endoplismic reticulumT. Stacks Of

smooth ER have been observed completely failing the center of the

haustorium. Between the cisternae of the sticied smooth erdoplasmic
reticulum very thin microtubules only 5O-100 A in diameter are

present (Fig. 66, 67). From nom on these microcubules will be

referred to as mini-microtubules, because of their smaller

diameter, as distinct from the normal microtubules. Mini-micro-

tubules appear to be thrown out to the periphery of the ER stacks

(Fig. 64). In many cases mini-microtubules have been seen in

bundles with or without ER associated with them (Fig. 65, 68).

They have also teen found tetween ER cisternae and the nuclear

envelope (Fig. 61), and smooth EP and plasmalemrira of the haustorium

(Fig. 60). Single mini-microtubules have not been seen. At higher

magnification these tubules appear to have subunits, most probably

six (Fig. 63, 69). Use of Markham et a!. (1963) image reinforcement

technique gives the strongest reinforcement at n = 6, n being the

number of equal arcs of a complete circle used in making multiple

exposures of the image. The mini-microtubules have been seen

running the entire length of the stacked smooth ER cisternae. They

appear to be a different organelle than the microtubules with 13

subunirs. The stacked smooth ER cuts off vesicles at its periphery

(Fig. 67). Acid phosphatase activity has been noticed over the

smooth ER stacks (Fig. 70).

The haustoria are packed with mitochondria which have plate-

like cristae. Some of the mitochondria have a peculiar shape, very

narro, in the middle a; if they are undergoing division (Fig. 53,

57). Some very long mitochondria have been observed (Fig. 55).

Microbodies have been seen in the vicinity of mitochondria (Fig. 63).

During the study an atypical haustorium was seen. This

haustorial mother cell had a large amount of rough endoplasmic

reticulum. Towards the periphery of the rough ER stacks, closed

spheres of rough E. appeared, with ribosomes inside (Fig. 51).

Between the vacuole and the ER, mitochondria could be seen. The

cytoplasm looked very diffuse and mitochondria stained differently

(Fig. 58). The plasmalemma of the haustorium appeared to be

invaginating at many places. The invaginations contained vesicles.

Free vesicles and multivesicular bodies were also present in the

cytoplasm. Thus, the haustorium had all the organelles that other

normal haustoria had except mini-microtubule;. This haustorium was

separated from the host protoplast by the host plasma membrane.

At tne si.- .

in the in:er-: -

is very tnir.,

The haustor.:-> r .:

substance : te=..:.

Two cntcr:, -

the body of ti.-

and Smith, Ir :- :

f.2,w;;" : :.e.,'" . :

that the per.t, :

accom-plis-ed i

case here. Th

is very sr.C.o o .

laminate of th, r.--

not penetrated t.

From theth a

of the ;r-" :._ .

general fungr l F.:,

1967, 1963; Coff-.

1963b, 1966, 1V-i.

1965; Litr.leficl

Peyton and Ecucrln.

cell that is c.r',-

iginjl infection, which normally lies deep

area of the host exoskeleton where the cuticle

inter of haustoria are ;ent by the mother cells.

rarely visible. There is a dark staining

:ises of adjacent haustoria (Fig. 54).

funi have been sho'in sending haustoria into

.-, .,F-rr c- S pp. on cockroaches (Richard;

.'-:ctr-:.J.2 r C...'t-.IoC:?arZ on lesser housefly

isler, 1968). Pichards and Smith suggested

Sthe tost in the case of &r "-.. ..: 7,_,, ; is

tic activity. The same appears to be the

through h ihich the neck of the haustorium runs

an and there appears to be no distortion of

cuticle. This suggests that the cuticle was

.iure alone.

--.rvatioi s it becomes clear that the haustoria

'-. Thaxter are not very different from the

l-a (Berlin and Bowen, 1964; Bracker, 1964,

.i:., 1912; Ehrlich and Ehrlich, 1962, 1963a,

S* ~~outh, 1956; Hardwick .- .22. 1971 ; Hawker,

Eracker, 1970, 1972; McKeen c. .2.., 1966;

. ). There is a thick walled haustorial mother

. ith the Rain body of the hauStorium through

a neck. The hau:.: r. is packed with nitochondria and endoplasmic

reticulumT, sg:: high rate of physiological activity. No

direct contact t-r. .;i the host protcplast and the h.austorium

exists. There is always a membrane that separates the two. This

membrane is continuous with the host plasma membrane.

However, in finer details the haustoria here are Yery different

from all others so far reported. The major difference is due to tile

structure of the host. The cells of termite epidermis, like other

animal cells, do not have rigid cell walls. The haustorial mother

cell is not in contact with the host cell at all. The neck in this

case passes through the integument of the termite and not the host

cell wall. The haustorium does not hang in the cytoplasm of many

epidermal cells. Thus the sheath around the haustorium is hounded

by the plasmalemma of as many cells as come in contact with it. As

reported above, in sone cases the host plasia membrane is very close

to the hausbrium while in others a space exists between the two and

in still others no contact between the two can be seen. The latter

condition is common in older haustoria. It appears as if with age

the haustoria push the epidermal cells aside and make room for

themselves. A similar situation has been reported for Kr.r-5yzs

spp., using the light microscope, where the basement membrane of the

integument has been seen bulging out into the body cavity (Richards

and Smith, 1956).

There fs some controversy and confusion concerning the nomen-

clature for structures associated with haustoria. I do not want

to add to this; therefore, I am not proposing any new terms. I

use the term haustorial sheath as was suggested by Etacker (1967)

and is presently being used by Bracker (1963), Littlefield and

Bracker (197G, 1972), and Coffey ct .:. (1972). However, I would

like to re-emphasize that haustoria of ...: "Lna :;;..:.: are not

sheathed like other haustcria and that unlike others (Littlefield

and Bracker, 1970) the neck is not Dsunded by an extrahaustorial

membrane. The collar is occasionally present at the base of the

haustorium but unlike other hau:toria does not surround the neck.

A dark-staining ring of wall material occurring miidway

between proxinal and dirtal endr of the haustorial neck in sections

stained with uranyl acetate and po'-titained with lead citrate has

been reported for sc.eral rust fungi (Ehrlich and Ehrlich, 171;

Hardwick et al., 1971, Cc.ffe)' .:2., 1972; Littlefield and er.cker,

1972). It has been suggested that the neck ring repre-int an

abrupt transition from the wall of the penetration pe. to the wall

of the haustorium (Littlefield .and Bracker, 1972). I .aT unable

to see any such band in the wall of the hauzcorial neck.

The wall of the hustorium is continuous all around without

any interruptions. Channels e. tendinm through the haastorial wall

providing physical continuity between the haustorial protoplast and

the boundary of the sheath as observed by Ehrlich and Ehrlich in

.Pajccirz-i n dr.r.:i z trI-t:- (1963, 1971) have not been seen.

The haustoria of Zriu--:. a n.; -2~ ': are unique in having mini-

microtubules. Similar structures have not been observed in fungi or

in any other group of animals or plants. Ilewcomb (1969) has recently

reviewed the literature concerning plant microtubules. Ordinary

wicrotubules are 180-300 A in diameter. They have an electron

lucent core about 100 A in diameter b.-unded by an electron opaque

carte.x or 3i11 about 70 A chick. They are separated by a space

of about 200 A or more from Each other suggesting that each micro-

tubule may be surrounded by a specialized zone. They appear to be

rather rigid unbranched structures usually following a straight

path. The wall of plant iTiicrotubules consists of thirteen fila-

mentous subunits (Ledbe'ter and Porter, 1964).
0 0
Steer and Newcomb (169) h, e reported some 290 A and 560-660 A

tubules in bean leaf glands. They appear to be different from

typical microtubules cheni;cailly. The smaller tubules have been

observed connected to the endoplasmic reticulum and they first

appeared in the perinucl-ar c3:oplasm between the layers of endo-

plasmic reticulun. llini-nicrotubules bear closest resemblance to

the tubules of P-protein bodies found in the phloem of the higher

plants. Cronshaw and Esau (1967, 1968) observed tubules in the

P-protein bodies of the sieve elements of uc.arbI;'ta nanmna and

Nicotiza, abac:,.-. They called the tubular protein P1-protein.
3 3
The tubules were 231 A in diameter in ,. tc:5bice and 242 A in

C. raUxir.. They also found fibrillar protein in the P-protein

bodies that replaced the tubular protein and assumed that the

tubular form becomes reorganized into the fibrillar form. The

fibrillar form of the protein designated as P2-protein. The

Pl-protein tubules appear to have sub-units and have a central

non-staining core. Parthasar.thy and Mijhlethaler (1969) reported

that in N. crba..c:e the protein tubule wall consists of 6 nearly

spherical units of 0O-70 A. The mini-microtubules are much smaller

in diameter; only 80-100 A across. They also have a central

nonstaining core and peripheral subunits. The number of subunits

is w it probably si,. Outside the haustorium in the snea:h of older

haustoria a fibrillar material resembling the P2-protein or tr:-

phloem is present. The mini-microtJtules are most commonly foaind

between the tubular cisternae of stacked smooth endopla:ici: reticulum.

They have, however, been seen in bundles in the general cytoplasmi

also. The cisternae of the stacked :Tmooth ER are continuous .:ith

the cisternae of the rough ER. I propose that the .roat'inr are

synthesized in the rough EF with the aid of ribosomnes in are

transferred to the smooth ER. These proteins are Fpol:i'rizcd and

mini-r.icrotubules are thus formed between, the cistern- of ti.e

stacked smooth ER. The newly formed raini-microtubules arr f.orceJ

out to the periphery and later In:o the general cytoplas-i. Fibrillar

material in the sheath of the haustorium might be the product of

depolyirerization of mini-microtubule protein, suggesting soire kind

of exchange between the host and the sheath.

The one atypical haustorium of r-i:' i. an-i.-: ,huich I

observed is very different from those of other fungal parasites.

Its protoplast is diffuse. Unlike other sister haujtoria it does not

have mini-microtubules. It looks as if it is being e3ten. The

increase in the number of inuaginations of the pladmIalere. iich are

unlike may represent sone kind of increased

activity there. These invaginations may iTmark some kind of pinocytotic

activity. Wheeler ;: :'. (1972) have recently reported pirccytosi;

in root cap cells. Pinocytosis on the host parasite interface has

been proposed by Ehrlich and Ehrlich (19E- ) and Berlin and Coten

(1964). Other workers have also reported aberrant h'ustoria

(Berlin and Boven, 1964; Littlefield and Bracker, 1972). The

necrotic haustoria were completely walled off from the host cyto-

plasm by a material continuous with the host cell wall. In termites

there are no cell walls sn tie aberrant haustorium might be cut

off by a physiological barrier that results from the host activity.

The other departure from normal in Zrmitaria : ,eri haustoria

is the presence of lysosomes that have not been reported so far in

other parasites. Autophagic vacuoles with mitochondria, ER, ribo-

somes and lipid droplets in them are commonly seen. There are other

big vacuoles with vesicles in them that exhibit acid-phosphatase

activity. The haustoria are packed with multivesicular bodies in

the vicinity of the lysosomes. The vesicles inside look similar

to the ones cut off on the periphery of the stacks of smooth ER

cisternae. Though these multihesicular bodies have not been seen

fusing with the lysosomes, they may be carrying lysosomal enzym-es.

It has already been suggested that the vacuolar system of plants

may represent the lysosomes, and that lysosomal enzymes are associated

with EP-derived microsomal vesicles (Matile, 1969). It is possible

that the haustoria of the erm.tar-ia .svneri are getting different

kinds of food from the termite than the haustoria inside a plant

host thereby making lysosomal activity necessary. By using Ei

autoradiography Ehrlich and Ehrlich (1970) have observed transfer

of 14 C from Pu.-nia' y-rmiz. :rstiei uredospores to wheat host


The haustoria of Ter:t- r s :,-ri are the first of its

kind studied. More work is needed to ascertain whether this


type of hustorial structure is unique or is shared with other

external ento-agenou parasi:es. Further study of the haustoria

of this fungus is also required to '.now iore about the nature of

mini-microtubules and of parasitism in general.


Figure 50. A thin section of old haustorial mother cell
X20,000. Notice the many-layered inner wall
(iw), smooth endoplasmic reticulum (er), lipid
droplets (L), mitochondria (M) and nucleus (N)
with nembranou2 structure inside the nucleoplasm

Figure 51. The rough endoplasmic reticulum (ER) of the
atypical haustorial mother cell 774,000. Arrows
point to the spheres of rough endoplasmic reticulum
with ribosores inside.

.... ... m..:: E ... "
B~~.;;i,,.~ ~~........ .: :
SF ^A -s
*3, ^



Figure 52. A median section through the neck of the haustorium
(n) X24,500. Notice the thich outer layer (ow) of
haustorial mother cell wall and the continuity of
the inner layer (iw) of the haustorial mother cell
wall with the inner layer (l) of the wall of the
haustorium. ilitochondrion (M), bundle of mini-
microtubules (DlT) and nicrotubules (MIT) are
present in the neck.

Figure 53. Near median section through a penetration site X20,750.
Haustorial mother cell (H.C) Is connected to the
haustorium (H) by a neck (n). Outer layer of
haustorial mother cell wall (oW) and that of
haustorial wall (ON) are dark while the inner
layers of both haustorial mother cell wall (IW)
and haustorial wall (IW) are electron light and
the two appear to be continuous. The hole in the
cuticle (CU) through which the neck runs appears
to be very smooth. Nucleus (rN) has a nucleolus
(NU) and is bounded by a double membraned nuclear
envelope (NE). Certain miembranous structures are
present in the nucleoalasm (arrows). Open arrows
point to the constricted mitcchordria.

* - '! ~


g .


Figure 54. Thin section of old hausto-ia (H) at the site of
original infection Y3,20'iC. Hautoria have lysorres
(1) and nucleus and are separated from the host
protoplast (h) by a sheath (S).

Figure 55. Thin section of hausteria (H) underneath the cuticle
(CU) of the termite X7,200. HausLoria are separated
from the host protoplast (h) by a sheath (S). Notice
the enormous size of the mitochondria (11).

Figure 56. Thin section of the hauscorium X.35,750. Plasma
membrane is connected (arrow) to a plasrialemmasome
(PL) projecting into the haustorial cytoplasm.
Outside the haustoriu. in the sheath there is
fibrillar material (fm). A microtubule (IIT) is
present inside the haustorium cytoplasm.

Figure 57. Haustorial mitochondrion constricted in the middle
X82,000. Arrow points to the connection between
the crista and the inner mitochondrial rmerbrane.
Notice the nearby mini-microtubules (MI1T) and
rough endoplasiric reticulum (ER).

ira -~i I ., k


Figure 58. A portion of a typical hau.torium with aberrant
mitochondria X35,750. Protoplasm of the haustorium
looks diffuse. Plasma meiTi brare is inv'aginated at
many places (arrows). The invaginations have
vesicles in then. Few vehicles are present in the
cytoplasm. Uall of the haustorium (WI) has two
distinct layers. Host protoplasm (h) is also in

Figure 59. Host-parasite interface 82,000. The host plasma
membrane (hPM) tightly fits against the haustorium
wall (U). Compare the diameter of mini-microtubules
(MIIT) of the haustoriun with that of the micro-
tubules (MT) of the host cytoplasm (hi.

Figure 60. Transversely cut mini-microtubules between the
plasr.a membrane (P;I) of the haustroium and its
endoplasmic reticulum (ER). Wall of the haustorium
(W) is .'ery thick.

Figure 61. flini-microtubule running (arrow) between nuclear
envelope (NiE) and the endoplasmic reticulum-

Figure 62. Periodic acid silver-stained haustoria ,43,750.
Haustorial wall (W) is PAS-positive while fibrillar
material (fm) in the sheath is PAS-negative.

Figure 63. Ilicrobody (ME) of the haustorium X54,500. Notice
the close association with the endoplasmic reticulum
(ER). Mlitochondrion (M) is also in vicinity.

aB J

.- .1 -. I I .

I .. .., i8

,<. *- .- i..
L-. .. .. r I "' "



-. ,
V .-:-. :/ ;. '*-*


Figure 64. lini-microtubules (r-IT) between the cisternae of
endoplasmic reticulum (ER) .109,000. The mini-
microtubules appear to be produced by endoplasmic
reticulum and extruded towards the periphery.

Figure 65. A bundle of longitudinally cut mini-microtubules

Figure 66. A stack of endoplasmic reticulum with longitudinally
cut mini-micratubules between the cisternae (arrows)

Figure 67. A stack of endoplasmic reticulum with transversely
cut mini-microtubules between the cisternae X30,000.
The endoplasmic reticulum is cutting off vehicles
(arrow) at the periphery. Certain vesicles (VS)
are present in the cytoplasm.

Figure 68. A bundle of mini-nicrotubules cut transversely

Figure 69. Mini-microtubules cut transversely X824,000. Six
subunits are clear (arrow) in the viall of the
mini-microtubule. Compare the endoplasmic reticulum
membrane with the subunits of the wall of mini-




Figure 70. Acid phosphatase activity in the hauscorium
X56,000. The activity can be seen in the lysomer
(1) and over the steck of smooth endoplasnic
reticulun (er) cisternae with mini-microtubules
between then. The mitochondria (I) and the
haustorial wall (W) show no such activity.

Figure 71. A lipid droplet being surrounded by endoplasmic
reticulum forming autophagic vacuole .51,500.

Figure 72. Autophjgic vacuole (1) with mitochondrion (H)
inside X51,500. The wall of the haustorium (U)
and the host protoplasm (h) are also seen.

Figure 73. Haustorium with lysosome (1), mitochondrion (1)
and multivesicular bodies (rlVB) '53,000. Vehicles
can also be seen inside the lysosome.


j".4 '',^". ^' -S-r
,"fc "- '.*' 'l *

.C ~1 r


c .



When Tha>ter (1920) described ?er-itri.c as a new genus of

entomophilous fungi on termites, he visualized it as a closely

compacted sporodochium and suggested that "its mature condition,

however, evidently a Fungus Imperfectus, seems to give it a formal

place among the Leptostromaceae." Others (Reichensperger, 1923;

Feytaud and Dieu:eide, 1927; Colla, 1929; Heim et 2:., 1951) also

studied this genus but only Colla (1929) and Heim (personal commu-

nication) had a different opinion about its taxonomic position. Colla

suggested that a new family should be created and Heim thinks it

belongs in Tuberculariales of Fungi Imperfecti. My studies (Chapters

I, II, Ill) have shown a number of features of Te'r-ircar a that were

not reported by these authors. These include the ecto-parasitic

nature of the fungus, the so-called bhlamydospores being haustorial

mother cells in reality, sending haustoria into the host integument,

and the phialoconidial nature of the spores. These discoveries

necessitate an emendation of the genus.

ernita ri: Thaxt. emend. Khan

Entomophilous; primary thallus crust-like, stromatic, few cells thick,

variously shaped with even margin; haustoria penetrate the integument

from specialized thick-walled haustorial mother cells; whole primary

thallus matures into a sporodochium of tightly held phialides making

a hymenium over a basal pseljdoparenchrymtous subhymenium, with a

peripheral excipulum of black thick-walled, sterile cells, margins

lichenoid, spreading but even; phialides cut off conidia apically

in basipetal succession at conidiocenoiu loci into distinct collar-

ettes; collarettes long containing four or more conidia; conidia

catenate, 3.5 4 u X 1.5 2 p, cylindrical, truncate or rounded

at ends, hyaline in mass.

Thaxter originally proposed two species, T. snyderi and T.

corc,:::c, on the basis of the length of the phialides, the length

of the collarettes and the structure of the tip of the phialide.

The phialides and collarettes of .;r--:.:,r ca~ro..a-z are larger in

diameter and length than those of T. ,kI-'r. The phialide tip of

T. n'siri is blunt and that of ;. Joor, r: has minute pointed

prolongations resulting in a minute, echinulate hynenial surface.

Later, Reichensperger (1923) described 2 -r-ni:cia from the Belgian

Congo and Brazil and proposed a new name .. t;ku--rr. for the Brazilian

specimen "should it turn out to be a new species." Having not

examined the type it is impossible for me to know whether it was a

new species. Later. Colla (1929) reported the presence of ;.

,3':.ri and 2. corP.ira, the former from Chile and Brazil and the

latter from British Guayana, Costa Rica and Phillipines, growing

on termites' exoskelecons.

Colla (1929) also described a new genus from British Gua/ana,

parasitizing the integument of the termite ilcre-e r.~'::~a =

(L) and proposed a new genus ;a:ciazt l!iz with a single species

M. sig:s:ri. .at::rol-lZa closely resembles TerSitri'a but its

sporodochium has 8 to 10 special cavities containing fertile hyphae

surrounded by sterile ones. Colla described 'I. silv stri? as having

a star-shaped thallus in young as well as adult stages, the arms

being intensely black, carbonaceous and sterile. The mature thallus

which is a sporodochium 60-70 u in thickness has three distinct

regions, a basal 12-20 p thick subh;-inium, a middle 37-40 u thick

hymenium, and an apical epihjirenium. The basal layer of the

pseudoparenchyTratous subhirieniui is comprised of thick walled

cells. A foot is sent into the integunent which according to

Colla (1929) reacts with maximum hypertrophy of the epidermal cells.

Thaxter (1920) reported a similar response to ?Ter?.t:: a infection

by the epidermal cells of the termite integument. Through my

studies I know that what ThaAter thought were hypertrophied

epidermal cells actually were haustoria of the Teriit.ria. It can

be assumed from Colla's figures that in the case of ,-cctircleaZ

too, those hpertrophied epidermal cells actually here haustoria.

Thus, in the basal layer of the subhymenium there are certain

specialized haustorial mother cells that send haustoria into the

integument. The hymenium is loculate, locules being separated from

each other by the columns of sterile hyphae that run from the sub-

hymenium upward, anstamose at the top of the hymenium and form an

epihymenium. These locules are full of what Colla called fruiting

bodies. She thought they resembled asci. However, shewas not sure

of this nor was shesure of the phialidic nature of the conidiogenous

cells of Territ.ria. She described them as having spores like that

of 7tr-.;r-5 : but smaller in size. She also desc:rited a dark line

across the sporogenous cells at about two-thir.-; of their length.

This dark line resentles the dark line across the hymenium of the

Termitaria that marks the position of the conidiogenous loi of the

phialides. I think that the fruiting bodies were actually phialides

and as Colla herself said it was not possible for her to confirm the

nature of these sporogenous structures because of their small size.

The epihyrenium is sterile and i.akes an arch over the phialides

in the locular areas.

During this study I had the goodJ fortune of looking at some of

the slides of r.;.r:c-'r; made by Tnadter (courtesy Farlow Her'barium).

These slides were in very good condition and I found that slide

nu-ber 3240i of iar -'.-i;a r:r.c.::- s not a : ,- tri but

t'.ittir~clUl The furigus was collected froni Panama and the slide

was made in 1924. The "lide has rany cross sections of the

sporocarp showing subhymenium, hymenlum, epihrmenriumii and the sterile

hyphae dividing the hymenium into r-ny chambers containing phialides

(Fig. 75). There is a basal layer of thick called cells many of

which are haustorial mother cells sending haustoria through the

cuticle ( 79). Over this there is a pseudoparench.,Tatcous

subhyr'enium 13 u in thickness from which ari;e the phialides

arranged in a palisade-like layer making a broad 52 u thick

hymenium. Tre hymenium is traversed by sterile hyphae that divide

it into chai kers. The apices of all the fertile hyphae (phialides)

and sterile nyphie are fused with each other and it appears that at

maturity the sterile hyphae elongate, as a result of which the thick

tips of the phialides break off (Fig. 78) and become a part of the

epihymenium. The broken ends of the phialides remain free in the

cavity under the eplhymenium. It is assumed that the conidia are

released in those cavities, put a pressure on the epihymenium that

is broken and flaked off. The epihymenium is 5 u thick and has a

reticulate surface (Fig. 80). The space between it and the broken

phialide tips is about 8 u thick. The conidiogenous loci of all the

phialides lie at their bases and each collarette which is approAimately

as long as the thickness of the hyrenium has 10-12 truncate, hyaline

conidia. The conidia are 2.5 3.0 u X 1.5 n, much smaller than that

of the Ti-itaric and broader compared to their size (Fig. 74). The

whole sporocarp is 85-90 u thick.

From the description of the fungus it becomes apparent that this

one is slightly different from :!. sirlestrii in having longer phialides,

thicker sporocarp, conidiogenous loci lying at the bases of the

phialides and that the surface of the epihymenium is reticulate in

this species. Colla's figures also show that the sterile hyphae

partitioning the hymenium into chambers make wider columns in

tU. siZest rii. Thus, it is proposed here that this fungus be

regarded as a new species:

MattirellZZ.: c -lsosa sp. nov. Figs. 74-80.

Entomogenous; thallus maturus sporodochium, densus 85-90 u;

hymenium densum 52 u divisum in receptacula numerosa colu.nis

hypharum sterilium latarum paucilocularibus; phialides strict,

loca conidiogenera sunt in fundamentis phialidum, apices crassi

phialidum et apices hypharum steriliur, sese conglutinanat epihymeniumi

formantes, prolon.jatio hypharuaTi sterilium phialidium apices frangunt,

cavitatem super apices fractos efficientes; conidii enodojeniter

format, 10-12 in coll retta singular, truncati, citenr ti, 2.3 3.0 .

1.5 u, hyalini et cylindrati; superficies epihymerni reticulatus.

Entonogenous; mature thallus a sporodochijum, 35-90 u thick;

hyaienium 52 u thick, divided into rany chambers b frew cells wide

columns of sterile hyphae; phialides tightly held, conidioglenous loci

lie at the bases of the phialides, tnick tips of phialijes and the

tips of sterile hjphae fuse with each other forming an epih,ieniun,

elongation of the sterile hyphae break the phialide tips mal ing

cavity over the broker tips, conidia formed endogenously, 10-12

in each collarette, truncate, catenate, 2.3 3.0 X 1.5 u, h-,aline

and cylindrical; the surface of the epihimenium reticulate.

uct:ircLoea and e ni:ria have iany characteristic: in common.

They both are entomophilous, sending haustoria from specialized

thick walled haustorial mother cells into the inte-gument through

the cuticle of the host, and the haustoria do not go beyond the

epidermis. Both have a foot-like structure nhich according to this

study of 'Te ..ii.a 'i .- .'f- marks the position of original infection

and penetration. Both have a crustose thallus which is stroTmatic in

young stages. In both cases the whole thallus matures into a

sporodochium that has a pseudoparenchynaatous subhymenium, and a

hjnenium of phialides. There is an excipulun around the sporodochium

in both genera. However, they differ in iany respects. All cells

in the basal layer of the thallus are thick walled in Mtti'r:Zejll

while in 7e. r.iar.ia only a select number are thickened. There is

an epihymenium present in L:i:zle21 while in :e- ::-".i there is

none. The 7ymenium of X:ti;rc:>EiZ is divided into many chambers

containing phialides separated from each other by the columns of

sterile hyphae.

On the basis of major difference; between the t.o, Colla (1929)

proposed the genus .larir,!;' .2 but suggested that it be put in a

single family. She did not suggest any name for the family.

The characters of the two genera are so unique that it is very

difficult to put them into any recognized group of Fungi Imperfecti.

That may be the reason why the taxonomic position of the two is

still undetermined.

The imperfect fungi are placed under subdivision Deuteromycotina

by Ainsworth (1971). The Deuteromycotina is subdivided as follows:


Blastomycetes -- yeast and yeast-like forms.

Hyphomycetes -- mycelial formTs with or without conidia.

Agonomycetales -- without conidia.

Hyphomycetales -- conidia not on synnemata or sporodochia.

Stilbellales -- synnemateous.

Tuberculariales -- sporodochial.

Coelomycetes -- conidia within acer'uli or pycnidia.

Sphaeropsidales -- pycnidial.

Melanconiales -- acervulial.

Two other orders of Fungi Imperfecti have been proposed,

Peltasterales and Gloeohaustoriales, by Batista and Ciferri (1959)

and Heim (1952) respectively, the former for the imperfect states of

Microtheriales that have pycnidia with inverted hymenium and the

litter for such entoogenou eno nera as 4:,; .'... 3oZ and

C;.;.tra,!c-T: r i Ob.'viousl. ".':: -rc.' j and ta-n'..:v, do not

belong to these to order;. They resemble only rmeiTiers of the

orders Sphaeropsidales or Tuber:ul.ariales because the sporocarp

of :-t.:.:,:-L ri is closer to the sporodochiur of tie latter while

that of .'2:s:ir-J, ;;.. is closer to the stron having many pycnidi

embedded in it like in Sphaeropsidales. The spo'rodochium i a

determinate, pulvinate masi of conidiophores tightly clustered on

a stromra 3'd ojo- not have an e-cipulum of fungal material. The

sporocarp of T?'-f i.'rf. develops on 3 stroratic primary thallus,

is comprised of tightly held phial ide, and has a peripheral e'cipulum

of black thick walled sterile cells. Thus, it 15 a modified form of

sporodochium. The pycnidiujm is defined as a variously shaped cavity

having a pseudoparenchniatous wall that is lined with conidiogenous

cells. It may be inmersed within a stroma or formed superficially

on the surface of the substratum. In ,.' ri'r-.:.::.w the h menial

cavities containing phialides can be superficially compared with the

pycnidia eurtedded in a strom,.

As has been discussed earlier 7ar-L-ia and !!:: .,,Z.rl appear

to be very closely related but differ in their sporocarp structure.

The two on the basis of their general morphology and the structure

of their sporocarps should not be put into any of the existing orders.

Therefore a new order is proposed here.

Termitariales order nov.

Entorogenous; ectoparasiticus, haustoria in integumentum

hospitis penetrans ex cellis matribus crassitunicatis specialibus;

haustoria restrict in texto epidennali hospitis; thallus principals

crustosus, stromaticus sine hyphis qui maturat in sporocarpo

perobscuro; sporocarpum coniritens subh)nenio pseudoparenchymate,

hymenio phialidibus haesis strictis et cincto e.cipulo sterili

cellarum crassitunicatarum; phialis cylindratus, phialonconidios

in ordine basipetalo in locis conidiigeneribus in collarettes

apicaliter abscindens, conidii eseptatis, hyalini, cylindrati,

catenati, marginibus truncatis aut rotundis.

Entomogenous; ectoparasitic, sending haustoria into the integument

of the host from specialized thick walled haustorial mother cells,

haustoria limited only to the epidermal tissue of the host; primary

thallus crustose, stromatic without any loose hjphae, matures into a

very black sporocarp; sporocarp consisting of a pseudoparenchymatous

subhymenium, a hyi enium of tightly held phialides and surrounded by

a sterile excipulum of tnick walled cells; phialides cylindrical,

cutting off phialoconodia apically in basipetal succession at conidio-

genous loci into collaret:es; conidia aseptate, hyaline, cylindrical,

catenate with truncate or round ends.

This is a provisional order created for Ter-i. sii and

-Stti rcJell.. Tentatively, a single family Termitariaceae will

be included, but when other entomogenous taxa are investigated or

perhaps sexual features of these genera are discovered, a complete

revision of these taxa will be necessary.

The phialides of 7...'-.':-,:': :d ,'L:'I.rcl .'a closely resemble

those of c--.-:'..7 rC r.::.: Eerk. ar.d Br. (Pirozynski and Mor.;an-Jones,

1968). C::- (Corda) F'penh., ,.';.KZ-:;~Z: S Peyron and Thielaviopsie

Went. The phialide: of all of them cut off chains of conidia in

basipetal succession at loci into the long collarettes.

The conidia are cylindrical, a:eptate and hyaline or lightly pigmenteJ.

Bloxamia belong: to Tuberculariales and i: saprophjtc uhile the

other three belong to H..phonyce.tals and can be found as -aprophytes.

Thil- .j;...f: is a serious: pathogen of higher plant:. The conldio-

phores of trm synnematio'us fungus E:*.'r'c:.; s Subram. also sho.a

some reserrbTance to the philides o r" -t-.r;,- ,iand '::: .'.;.:.l? ..

They are grzsjped in a single synnewj cutting off aseptate, hyaline

conidia erl' encusly at conidiog-n'ous loci in small collarettes.

The conidia are e.truded in basipetal succession through the open

ends of collarettes. The sporocarps of .n:r-I,-rt.I and 5:.:: ;'..z are

alike in .iiany respects. They are black, comrprised of tightly held

phialidjes whose conidiogenous J clci lie at the same distance from the

base of the phialides making a distinct line running across the


It can be said chat the members of the proposed order closely

resemble ipembers of the orders of class H)phomiycetes and therefore

it should be placed under the same class ne.t to Tuberculariales.


Figures 74-80. Mictit cKl, Ua .T:.ttC& sporocarp.

Figure 74. A portion of tne cro;s section of the sporocarp show-
ing subhyrenium (SH), h)yenium (HY), the zone of
conidiogenous loci (CL) and the conidia (C) inside
the collarettes X1,250.

Figure 75. Cross section of the sporocarp X250. The sporocarp
has three distinct regions, epihTnenium (EH). hyrenium
(HY), and subhm-nenium (SH). Columns of sterile hyphae
(ST) divide the hymenium into many chambers. Conidio-
genous loci (CL) at the bases of the phialides rake
a distinct zone. Basal layer (EL) and the excipulum
(E) are also seen.

Figure 76. Cross section of the sporocarp 1,000. Column of the
sterile hyphae (STJ divide the hmenium. Notice the
cavities (cv) over tUe phialides.

Figure 77. Cross section of the sporocarp X320. A column of
sterile hyphae (arrod) is seen all the wa) through
the hir.enium running between the epihymenium (EH)
and the basal layer (BL).

Figure 78. Cross section of the sporocarp K.1,000. The broken
tips of the phialides (arrovs) that fuse to help
make the epihymreniun (EH).

Figure 79. Cross section of the basal lajer of the sporocarp
X1,250. Thick walled haustorial mother cells (hr c)
send haustoria into the integument of the termite
through the channels (arrows) in the host cuticle

Figure 80.

Surface of the epihywnium X1,000. Note its
reticulate nature.

, :h



~-1' ~ ( 80i


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