DdLss.
Utrecht

1927

TER VERKRIJGING VAN DEN GRAAD VAN
DOCTOR IN DE WIS- EN NATUURKUNDE
AAN DE RIJKS-UNIVERSITEIT TE UTRECHT,
OP GEZAG VAN DEN RECTOR MAGNIFICUS
Dr. A. NOORDTZIJ, HOOGLEERAAR IN DE
FACULTEIT DER GODGELEERDHEID,
VOLGENS BESLUIT VAN DEN SENAAT DER
UNIVERSITEIT TEGEN DE BEDENKINGEN
VAN DE FACULTEIT DER WIS- EN NATUUR-
KUNDE TE VERDEDIGEN, OP DINSDAG
22 MAART 1927, DES NAMIDDAGS VIER UUR

• .. tr\' . • .

. A

Het is mij een aangename plicht, op deze plaats mijn hartelijken
dank te brengen aan allen, die mij gedurende mijn studietijd met
raad en daad hebben bijgestaan.

U, Hooggeleerde Sluiter, W ibaut, Sissing h, Du-
bois, Pannekoek en Zeergeleerde Büchner, ben ik zeer
erkentelijk voor alles, wat ik gedurende mijn eerste studiejaren van
U heb mogen leeren.

De persoonlijkheid van wijlen Professor Verschaffeit
heeft niet nagelaten ook op ons, jongere studenten, een diepen
indruk te maken. In dankbare herinnering denken wij allen aan
hem terug.

Hooggeleerde Weevers, de welwillendheid, die gij mij be-
toond hebt in den korten tijd dat ik met U samenwerkte, heb ik
zeer gewaardeerd.

Hooggeleerde de Meyere, het enthousiasme, dat gij voor Uw
vak gevoelt, heeft mij steeds met bewondering vervuld. Ik ben
U zeer erkentelijk voor de belangstelling, die gij in mijn werk hebt
gesteld.

Hooggeleerde Stomps, aan U heb ik veel te danken. Uw
colleges boeiden mij in hooge mate en hebben mijn liefde voor de
botanie aangewakkerd. De herinneringen, die ik bewaar aan de
onder Uw leiding gemaakte excursies naar Wimereux en Zuid-
Limburg behooren tot de prettigste uit mijn studententijd.

Hooggeleerde W e s t e r d ij k, hooggeachte promotrix, het is
mij niet mogelijk hier uit te spreken, hoeveel ik U verschuldigd
ben. Het heeft mij nooit berouwd, dat ik de Amsterdamsche Uni-
versiteit heb verlaten om in Baarn de Phytopathologie te gaan be-
oefenen. Het is voornamelijk aan Uw bezielende leiding te dan-
ken, dat het phytopathologische werk mij zooveel bevrediging
schenkt. Ik acht het een bijzonder voorrecht, dat ik dit werk nog
eenigen tijd in Baarn mag voortzetten.

Het bestuur van de stichting Willie Commelin Scholten ben ik
zeer erkentelijk voor de gastvrijheid, die ik op het laboratorium
heb genoten.

Ook den directeur van Cantonspark, professor P u 11 e, ben ik
veel dank verschuldigd voor de ruime mate, waarin ik steeds van
dezen hortus gebruik heb mogen maken.

Den Heer A. vanLuyk zeg ik dank voor de vele goede raad-
gevingen, die ik van hem mocht ontvangen tijdens de bewerking
van dit proefschrift. In het bijzonder bij het vervaardigen van de
foto\'s was zijn hulp onontbeerlijk.

Ook met den Heer J. G o o s s e n, hortulanus van Cantons-
park, heb ik op zeer aangename wijze samengewerkt.

Zeer dankbaar ben ik ook mijn huisgenoote Greta Mes, die
de vertaling van dit proefschrift, een lastig en ondankbaar werk,
op zich heeft genomen.

Ik waardeer het verder zeer, dat Mej. J. W ever zoo vrien-
delijk is geweest, het manuscript voor mij te tikken.

De welwillendheid, die ik van verschillende kanten ondervond
bij het verzamelen van materiaal, heb ik op hoogen prijs gesteld.

These investigations were originally undertaken with the idea
of adding something to our knowledge of the root rot of peas
caused by Phycomycetes. In America several publications have
appeared about it during the last few years, while very little at-
tention has up to now been given to it in Europe. It would, for
instance, be of importance to investigate if the dangerous ameri-
can root parasite of peas, Aphanomyces euteiches, Drechsler,
is also present in diseased pea roots in this country and if it is the
cause of rootrot.

As it was necessary that I first gained some experience with
Phycomycetes, I examined several different diseased plants which
were given to me as probably being infected by a fungus of that
group.

I will also discuss some of these diseases which up till now
were still unknown.

This applies in the first instance to the disease of the roots of
the Calla-lily which causes a considerable loss to the Ca//a-nur-
series in this country and has been thought to be due to Nema-
todes. It happened that diseased Chrysanthemum cuttings, which
had suddenly stopped growing, were also sent to the laboratory
at the time.

In the course of time I could examine different rots of cuttings,
several of which I described in this publication.

While examining the different pea roots it struck me, that
Asterocystis radicis d e W i 1 d e m a n, the wellknown parasite
on flax roots which is taken to be the cause of „brand" = fire in
flax, is often found on them. In order to be able to recognize this
Chytridiacea and not to mistake it for oogonia of Phycomycetes
I also examined roots of diseased flax. In doing so I discovered a

Phycomycete which is apparently parasitic on the flax roots, and
which I have therefore also described here.

As most of these plants had to be cultivated in vegetable mould it
was necessary to know something about the Phycomycetes which
inhabit humus. If our soil proved to be strongly infected by them,
inoculation experiments in it would be worthless. Some more
observations about the different Phycomycetes which I isolated
from seedlings grown in our vegetable mould will also have to be
considered.

This work was carried out more along phytopathological than
along morphological lines. I could only make a choice out of all
the isolations which were at my disposition.

A thorough treatment of the genera Pythium and Phytophthora
would lead me too far, so I am restricting myself to some conside-
rations about the classification of the group to which they belong,
and about some related genera.

In the year 1881 deBary(4) already wrote about the classifi-
cation of the group Phytophthoreae, in connection with the genera
Pythium and Phytophthora. He showed that there is far too little
difference between these genera to justify placing them in diffe-
rent families as Pringsheim had done (Pythium with the
Saprolegniaceae, Phytophth ora with the Pevonospotaceae). De
Bary pointed out, that there is really no other difference between
Pythium and Phytophthora than the way in which the zoospores
are formed. In the genus Pythium the protoplasm from the spo-
rangium flows into a vesicle in which the zoospores are then for- .
med, whereas in the genus Phytophthora the zoospores are formed
in the sporangium itself.

B u 11 er s monograph (10) about the genus Pythium is the best
source of data at present. He agrees with de Bary and also sees
no other difference between Pythium and Phytophthora. He in-
cludes both genera in the Peronosporaceae.

A detailed discussion about this question is found in a chapter
from „Generic concepts in the Pythiaceae and Blastocladiaceae" by
F i t z p a t r i c k ( 14), i. e. The Pythium — Phytophthora Problem.

This shows, that an absolute separation between Pythium and
Phytophthora is theoretically impossible, as it has become evident
that the traditional characteristic which separates Pythium from
Phytophthora, namely the formation of zoospores in a vesicle, in-

stead of in the sporangium, is also found amongst different Phy-
tophthora species. —

But in literature we find other species which belong to the group
of Pythium and Phytophthora. How are they related to Pythium
and Phytophthora?

In the first place the genus Pythiacystis. Barrett (2), E.
Smith and R. Smith (33) and Leonian(20) pointed out,
that a separation between Pythiacystis and Phytophthora cannot be
carried through. Smith and Smith even isolated forms, which showed
a transition from Pythiacystis to Phytophthora cactorum. Leonian
has now definitely dropped the genus Pythiacystis and uses the
name Phytophthora citrophthora.

Dufrenoy(13) includes the genus Blepharospora Petri in
the homogenous group of Phytophthora. Pythium and Pythiacystis
(which he, like Fitzpatrick(14), separates from the real Pero-
nosporaceae with aerial conidia and which are obligate parasites).

Fitzpatrick further regards the genus Pythiogeton v.
M i n d e n as a representative of this group.

According to my opinion, Pythiomorpha
Petersen is another genus which belongs
to it.

The question is, whether there are suffi-
cient reasons for letting these genera exist next
to Pythium and Phytophthora. I do not think,
that there ought to be any difficulty in let-
ting the genus Pythiogeton remain, as it is
sufficiently characterized by its asymetrical
sporangia. — It is a different question with
Blepharospora and Pythiomorpha however. —
According to my opinion these genera,
each with one representative, Blepharospora
cambivora Petri and Pythiomorpha gona-
podyides Petersen, should be included
in the genus Phytophthora. The reasons for
this I will give below.

I do not think that Petri (27, 28, 29)
succeeded in proving definitely that his
Blepharospora is not a Phytophthora.

1. that Blepharospora does not produce sporangia on the
surface of the infected parts of the plants or on solid cul-
ture media, but only in a mineral salt solution.

2. that Blepharospora possesses long, unbranched sporangio-
phores ending in one sporangium.

It will become obvious below that representatives of the genus
Phytophthora also show similar characteristics.

Blepharospora is related to P. cactomm, P. fagi and P.synngae,
according to Petri, because it possesses paragynous antheridia.
Although Pethybridge does no longer consider the posses-
sion of paragynous or amphigynous antheridia a reason for sub-
dividing the genus Phytophthora, as both kinds of antheridia
sometimes occur in one form, Petri still maintains that this sub-
division should remain.

A Phytophthora would then belong to the one or to the other
group, as it more regularly produces amphigynous or paragynous
antheridia. In this case Blepharospora would then belong close
to Phytophthora cactorum. I do not attach very much importance
to this production of antheridia, as Blepharospora forms oogonia
only in the tissues of the infected chestnut-tree, and it is therefore

difficult to determine the position of the antheridium. The charac-
teristics show that Blepharospora is more closely related to Phy-
tophthora cryptogea and P. cinnamomi.

These characteristics are exactly the same as the ones, Petri
uses to separate Blepharospora from the genus Phytophthora,
namely the difficulty in forming sporangia and the unbranched
sporangiophores.

Phytophthora cryptogea and P. cinnamomi also rarely produce
sporangia and possess little branched conidiophores. Further
more these two genera also have another characteristic in which
they correspond with Blepharospora. The sporangiophore grows
into the sporangium after its contents have been discharged. The
sporangia are also rounded off above, without a definite papilla
being formed, exactly as is the case with Blepharospora.

Petri separates Blepharospora from the genus Pythium because
the zoospores are already formed in the sporangium, the same
reason for which Phytophthora is separated from Pythium.

I therefore think that it is absolutely unjustified to let the genus
Blepharospora exist next to the genus Phytophthora.

The same counts for the genus Pythiomorpha established in 1910
by Petersen (25). Von Minden (22) already pointed out
that Petersen does not express himself about the reasons which in-
duced him to form even a new family, Pythiomorphaceae, but he
thinks Petersen is right. Pythiomorpha is again separated from
Pythium, because the zoospores are directly formed in the spo-
rangium instead of in a vesicle. The occurrence on loose twigs
and fruit in water is not a special characteristic of the genus
Phytophthora, but this is no reason for excluding Pythiomorpha
from the genus Phytophthora, as it is known (see Dufrenoy) (13)
that Phytophthora\'s may lead a saprophytic life.

In the detailed description of Pythiomorpha gonapodyides, the
only existing species of the genus Pythiomorpha, by Miss
Kanouse (19) there is no characteristic to be found which should
separate Pythiomorpha from the Phytophthora\'s. Petersen em-
phasized the fact that the mycelium is coloured purple by chloro-
jodide <of zinc, but when I applied the same method to Blepha-
rospora, it gave the same results.

the sporangium after the swarmspores have been set free and
sporangia and oogonia are formed with difficulty just as in Blepha-
rospora.

I think that I have now shown that the genera Blepharospora
and Pythiomorpha should be included in the genus Phytophthora,
so that we may speak of Phytophthora cambivora and Phytoph-
thora gonapodyides. Then except Pythiogeton, only the genera
Pythium and Phytophthora remain, as Pythiacystis has already
been included in the genus Phytophthora.

According to my opinion it would be of no practical importance
to unite these two genera into one. The name Phytophthora is so
universally used that it would be confusing to drop it in favour
of the (older) name Pythium.

It is enough to state, as other investigators have already done,
that theoretically no sharp line can be drawn between Pythium
and Phytophthora. In practice it is usually possible to determine
if the form which is examined belongs to the „type" Phytoph-
thora or to the „type" Pythium.

It becomes more difficult when no swarmspores are produced
as in the case of Pythium splendens Braun. Braun(8) does not
say if this fungus might perhaps be a Phytophthora. But as the
conidia of this form are all round instead of possessing the pear-
shaped aspect of most Phytophthora-sporangia, it was only reaso-
nable to speak of a Pythium instead of a Phytophthora, although
the shape of the spores is not an official difference. The same
will probably happen in other cases where difficulties are met
with in connection with the production of swarmspores.

Few genera have been studied so often as Phytophthora, and
yet the classification is far from being settled.

At present it is very difficult and sometimes nearly impossible,
to identify a Phytophthora. The different species of this genus
are so much alike, that it is nearly impossible to determine which
species one is dealing with. From the old species of d e Bary,
P. omnivora, the four species P. fagi, P. cactorum, P. syringae and
P.faberi have since been separated. Leon i an (20) now states

that there is no difference between P. fagi and P. cactorum, and
eliminates P. [agi.

There are many other difficulties in the same way. It was first
tried to settle these questions along purely morphological lines,
afterwards physiological methods were tried for the same purpose.

Rosenbaum (32) wrote a very accurate study in which he
gives a detailed morphological account of the different species. He
finally separates them by means of several characteristics, for in-
stance the difference in size of the zoosporangia and oogonia as
is indicated by curves.

But this is a very difficult method to apply in practice. This
publication of Rosenbaum is however still a very valuable acqui-
sition to the Phytophthora literature, especially as the species
are studied on different culture-media. Leonian\'s research was
meant to give a solution of the problem along physiological lines.
He declares, specially in connection with his investigations about
the mutations of Phytophthora parasitica rhei, that the genus may
not be classified according to the size of the sporangia, the pre-
sence or absence of oogonia, other characteristics which appear
in culture, and pathogenicity.

In practice there are however many objections against his
„physiological key". Leonian himself also realizes that it is very
difficult to cultivate fungi under exactly the same physiological
circumstances in different parts of the world.

It therefore becomes nearly impossible to split the genus up into
several divisions and to draw a sharp line round each. The matter
has become more complicated by the publications of A s h b y (1)
and Gadd (15).

They demonstrated, that heterothallism is found in the species
Phytophthora [aberi. This could also show, that different species
of Phytophthora may form hybrids in this way, and as transi-
tions between the different forms will then of course originate,
a sharp separation between them would become absolutely im-
possible.

According to my opinion, the morphological differences between
the species must for the present first be determined and then
physiological characteristics may be added, so that we may get
a system which will be useful in practice. The more so, as it is

easier to identify by means of morphological characteristics, than
by the round about physiological method.

In the genus Pythium things are not so complicated as with
Phytophthora. Still it is not at all easy to determine if a special
isolation belongs to the otherwise wellknown Pythium de Baryanum
or not.

Even Drechsler(12) who is writing a monograph about the
genus Pythium speaks of „species", or „forms" of the de Barya-
num type. I think that we must now be specially careful not to
give too strict definitions of the species, as the Phycomycetes are
very variable forms. Some species of Pythium for instance show
a different tendency towards forming oogonia or sporangia. D e
Bary (4) already noticed this in 1881 with P. megalacanthum.
I possess only one isolation of this species which forms only
oogonia and no sporangia.

The same is the case with an undescribed Pythium I isolated
from various plants. The one isolation forms sporangia and swarm-
spores, although only few, the other nearly purely oogonia.

Nothing further is known about the sexuality of these forms. —
Perhaps the many forms one meets without oogonia show that
heterothallism exists as in P. faberi, or perhaps we have here a
strong tendency towards production of asexual spores. This ap-
pears to be the case with P.de Baryanum var, geranii Braun(8)
which produced a few oogonia in culture only after 14 months
which even then often degenerated.

I never noticed anything of biological races adapted to certain
hosts. According to Drechsler however, some forms of Pythium
de Baryanum are more pathogenic than other forms of the same
species.

This again shows the great variability which exists, also in
connection which physiological characteristics. It is therefore ad-
visable not to give too strict a. definition of the different species.

In making isolations from infected roots of the different hosts
I proceeded as follows:

The diseased roots were layed out on agarplates without being
disinfected, as this handling would have killed the delicate myce-
lium of the Phycomycetes. At first I always used cherry-agar for
the isolations as it is rather acid and therefore arrests growth
of bacteria. Afterwards I found that oatmeal-agar is a more desi-
rable medium. Although it is more favourable for bacterial growth,
the Phycomycetes grow better on it than on cherry-agar.

It is very difficult to keep the cultures absolutely free from
bacteria as they adhere to the hyphae and are therefore difficult
to remove. The best way is to cultivate the fungi on agarplates
and to transfer them as often as possible.

I usually used oatmeal-agar for keeping them in culture. On
cherry-agar they mostly also produce a woolly growth of mycelium
but few oogonia are formed. Pythium de Baryanum however grows
well on cherry-agar while P. megalacanthum does not grow at all.

On cornmeal-agar many oogonia are also formed, but this
medium is even more favourable for the growth of bacteria and
therefore not so suitable.

Swarmsporangia are, on a whole, seldom produced on agar
media. In order to obtain them I. adopted the method used by
Petri for Blepharospora cambivora.

I cultivated a tuft of the mycelium from agarcultures for a few
days in a flask, in which a thin layer of this solution had been
brought. Hardly any growth of mycelium took place but apparently
this anorganic solution served as a stimulus for the production of
sporangia. The greatest number of sporangia were however for-
med when the bit of mycelium was afterwards transferred from
the solution to a watchglass with distilled water. Sometimes the
zoospores appeared directly; in other cases the water had to be
refreshed first, and zoospores only appeared afterwards, for in-
stance after one day. This method was successful with several
kinds of Phytophthora-and Pythium-species which had at first
hardly produced any zoospores, or none at all.

Even species which had been isolated years ago and had since
then been kept in culture at the „Centraal Bureau voor
Schimmelculture s", could be induced to form zoospores
when treated in this way. With Aphanomyces euteiches I also
obtained the same results.

It is a pity that the results are not always so satisfactory:
Pythium splendens for instance, cannot be induced to form zoo-
spores even by this method. In other cases there are so few that it
is impossible to trace their development.

For inoculation material I used some strains which I had iso-
lated from diseased plants, and some species which were kept in
culture at the „Centraal Bureau v. Schimmelcul-
ture s". I never found any indications that the pathogenicity of
these species had suffered by remaining in culture for so long.
Pythium complectens from B r a u n, Pythium splendens from
Leonian, and P. de Baryanum from Hartley which had
been cultivated for some time, were still very virulent.

I will enter more fully into the methods of infection when I
describe the experiments. The main point is that I often used
watercultures in which the fungus was brought (see chapters on
Calla-lily, peas and flax). As for the cuttings, their lower ends

Whenever I used vegetable mould for growing peas in, I steri-
lized it before use, because of the many Phycomycetes occurring
in humus. (See chapter on Occurrence of Pythium in vegetable
mould).

Large hothouses with Calla-lilies are found on several commer-
cial nurseries in our country. It is a very profitable culture as the
flowers are very valuable and as each hothouse may yield some
thousands of flowers.

In spring they are put in the open, so that they may dry out
and pass through a period of rest. After this period the corms
are taken out of the pots, cleaned and planted again in fresh
pots. They are left outside for another little while (not dry this
time) and put in the hothouses at the beginning of winter. They
start flowering in about December. In the last few years it often
happened at several nurseries that the plants started to fade in
December. The outer leaves then showed a yellow discoloration,
which slowly spread over the surface until the whole leaf had
turned yellow. The same happens to the other leaves until there
are only a few left. The flowers are few and small so that the
yield of a hothouse with diseased plants is very small. The disease
is not very acute as new leaves are still being formed and the
plant is not killed, but it remains ailing.

A diseased plant is easily withdrawn from the pots as the roots
are easily broken off, and only small rotten pieces still adhere to
the corm. The corm itself is sometimes also full of holes and grooves.
If the plant is very carefully taken out of the pot with soil and

all, the roots appear to be badly infected. They have a glassy
and drooping appearance, especially striking with the thicker roots.
A reddish discoloration is also often found.

If the plants are treated in the usual way, e. g. if they pass
through a period of rest and are then changed to other pots, there
is at first no evidence of the disease. But, about the flowering
season the symptoms appear again. It stands to reason that the
corms suffer badly from such repeated occurrence of the disease.

§ 2. Microscopical examination of the roots and isolation
of the pathogenic organism

One receives the same confused impression when examining the
infected roots of the Calla-lily as when examining other diseased
roots. First one notices the many Nematodes. The disease had
at first been ascribed to them, but as most of them belonged to
the not-parasitical kind Rhabditis brevispina, which lives in all
kinds of decaying portions of plants, their presence was still no
proof that they are indeed the cause of the disease.

Besides these Nematodes different kinds of soil-fungi are found
in the roots. Thielavia basicola was occasionally found and also
often oogonia of Phycomycetes. It was tried to find the cause by
laying out small pieces of infected roots on agarplates. As the
roots were so delicate, it was of course impossible to sterilize them
first. This proved to be a great objection as all kinds of fast
growing soil-fungi and saprophytes, which immediately attack
the decaying roots, now appeared in my cultures. The isolations
therefore only yielded common fungi such as Pythium-species and
Botrytis. They are just ordinary inhabitants of vegetable mould
and it was improbable that they would be the cause of the Calla-
lily disease, whicTi appeared relatively suddenly. After several
unsuccessful attempts I gave up this method of isolating the patho-
genic organism.

In the nurseries it had become evident that the disease also
appears when the corms were dried and planted in absolutely fresh
soil. This would show that the disease is transmitted by the corms.

It was therefore necessary to cultivate the corms under such
circumstances that ordinary soil-fungi would be excluded. If the

corms then still became infected, it ought to be easier to isolate
the pathogenic organism. As Calla-lilies under ordinary circum-
stances grow in marshes and swamps, it was probable that it
would be possible to cultivate them in culture-solutions. As the
big

I took some young corms of infected plants, brushed half the num-
ber with water and the other half with a fairly strong solution of
formalin (commercial solution diluted about 20 times). I then took
a glass vesicle which had first been painted black and then white,
so that the temperature of the liquid inside would not become too
high. A lid, through which a few holes had been made, could be
screwed on it. The vesicle was filled with culture solution of
V. d. Crone:

1 L. water
1 gr. KNOg
0,5 gr. Ca SO4
0,5 gr. Mg SO4
0,25 gr. Feg (P04)2
0,25 gr. Cag (P04)2.

The young corms were allowed to hang in the culture solution
and were fastened in the holes in the lid by means of two pieces
of cork, which fitted in each hole. In this way the leaves could
freely develop into the air while the corms touched the culture-
solution. After some time it became evident that the plants which
had been treated with formalin, had developed excellently. The
roots were strong and healthy. The roots of the plants which had
only been treated with water, on the other hand, soon showed
signs of infection. This rot could not be the result of an organism
present in the solution, as the formalin-plants remained healthy.

When the infected roots of the plants in the solution were
examined microscopically, they were found to be covered by a
large number of sporangia, which soon formed swarmspores in
water. Hardly any other fungi were present. When I layed out
small pieces of these infected roots on agarplates the fungus only
grew very slowly, although such a number of swarmsporangia
were present.

I nevertheless succeeded in isolating it in this way as the usual
soil-fungi were absent.

Inoculation experiments, which will be described below, prov-
ed, that the fungus with swarmsporangia which was found
on the diseased roots was indeed the cause of the rot. I afterwards
also found it on a root of a diseased plant which had not been
grown in waterculture but had been taken out of the soil.

The way in which the swarmspores are formed could directly
be followed when infected roots were examined in water. It ap-
peared that this happens inside the sporangia in the way charac-
teristic for Phytophthora.

It also became evident that, after the swarmspores have been
set free, the sporangiophore grows into the sporangium in the
same way as is found in the group of Phytophthora cryptogea, as
I have already pointed out.

Little branched sporangiophores and sporangia without a dis-
tinct germinal papilla are also found.

Many oogonia with amphigynous antheridia were present in
cuhures on oatmeal-agar, but sporangia never appeared. These
were only produced when I applied the method described by Petri.

The Phytophthora of the Calla-lily is closely related to P. cryp-
togea, P. cambivora, P. gonapodyides and P. cinnamomi.

It differs from P. cinnamomi in the absence of chlamydospores.
From the others it differs in the size of the oogonia. The oogonia
of the Phytophthora of the Calla-lily generally measure ± 34
38 fi, while the average of those of P. cryptogea is given
as 30 fi.

For the oospore of P. cambivora Petri gives measurements of
20—27 fi and for the oogonia of P. gonapodyides measurements
of 22—36 fi are given by Miss Kanouse.

Besides, while the Calla-Phytophthora forms oogonia abun-
dantly, P. gonapodyides and P. cambivora produce no oogonia in
culture and P. cryptogea only a few on oatmeal-agar.

Oogonia are no longer produced by an old culture of P. cryp-
togea present in the collection of the Centraal Bureau voor Schim-
melcultures. It was therefore impossible for me to compare the

oogonia of the Calla-Phytophthora with those of the three last
mentioned Phytophthora\'s. Nevertheless I think, that the colour
of the wall of the oogonium and oospore also differs from those
described by Pethybridge for P. cryptogea.

In this way there are various characteristics in which the Phy-
tophthora of the Calla differs from the forms closely related to it.

Also the special occurrence in the roots
of the Calla-lily and the virulent action
on them, made me decide to form a new
species Phytophthora Richardiae.

The mycelium is the same as that
of other Phycomycetes. Sporangia vary
in size as is apparent in the drawings.
They are produced by the method of
Petri, or by cultivating plants in water-
cultures and infecting the roots with
the fungus. Zoospores reniform, bici-
liate, ± 10 in size, produced in
varying numbers in one sporangium.
Oogonia produced in oatmeal-agar and in tissues of infected
roots, obovate, growing through antheridium, diameter of distal
end 34—38 Oogonial wall yellow. Oospores 29 ft in
diameter, wall 2—Z\'/g fi. in diameter and greenish purple in colour.

Antheridia with protuberances, amphigynous, occurring on other
hyphae than the oogonia.

Very virulent parasite of the roots of the Calla-lily causing
them to decay. May also penetrate into the corms with the
same effect.

It now had to be proved by inoculation experiments, that the
Phytophthora is indeed the cause of the above mentioned disease.
I first used small corms for these experiments because they are
easier to work with.

corms treated with formahn. They were quite healthy and grew
vigorously in the culture solution, although they had been taken
from diseased plants. Small pieces of agar with Phytophthora
mycelium were smeared on the roots. After a few days the roots
began to show signs of decay, they became flabby and took on a
glassy appearance.

This spread further and further until after some time all the
roots were badly infected.

In order to be absolutely sure, I then inoculated corms of healthy
plants which had also been brushed with formalin. These inocula-
tions gave the same results as the previous ones. The outer leaf
of an infected corm soon shows the same symptoms as appear in
fullgrown diseased plants.

Finally I also infected large corms before planting them in pots,
by rubbing a culture of Phytophthora on them. Corms treated in
this way formed very few roots and on a whole showed the same
symptoms of disease.

As the corms are sometimes only partly rotten, I wanted to find
out if this was also due to infection by Phytophthora. I therefore
laid slices of the corms in petridishes on wet filterpaper and in-
oculated them with the Phytophthora. A very peculiar rot, dif-
fering from the slimy bacterial rot, appeared. The cells seemed to
become detached from each other and a fairly dry mass was formed.

It was now necessary to find a method of controlling the disease.
As I had been successful in treating the corms with formalin it
was probable that a solution to the problem could be found in
this direction. Originally I had immersed the corms before plan-
ting them in a 2 o/q solution of formalin and then brushed them
with it.

As it appeared that this treatment was not sufficient, I think
it better to leave them in a weaker solution for some time. The
rotten places should be cut out.

Investigations of Bewley (5,6) already showed that the
corms do not suffer by this treatment with formalin. Bewley repor-
ted an epidemic in Calla-lily nurseries, which seems to be very

Two young corms of Calla after having grown for soma time in
V. d. Crone-cuUure-solution. They originated from a diseased plant,
but were brushed with formalin before being placed in the culture-
solution.

The two plants at the right originated from a diseased Calla and
were handled in the same way as the plants in plate 1.

The young corm at the left originated from a healthy Calla and was
also inoculated with Phytophthora Richardiae.

similar to the above described disease. He takes Bacillus aroideae
T o w n s e n d to be the cause, but does not describe any inocula-
tion experiments with this organism. Townsend (36) says about
the symptoms of his disease: „The writer found the Calla\'s rotting
off usually at or just below the surface of the ground, the disease
sometimes extending down into the corm, sometimes upward into
the leaves, and frequently in both directions. Occasionally the
disease seemed to start in the edge of the leafstalk, in the flower
stalk or in some underground part of the corm, though as a rule
it started at the top of the corm just above, but near the surface
of the ground".

These symptoms differ in many respects from the ones I noticed
in the Calla-nurseries in Holland.

Although there seems to be a great similarity between the symp-
toms described by Bewley, and those of the disease in Holland, I
am not able to say if the two diseases are identical, as I never had
the chance of examining Bewley\'s specimens.

Bewley used a solution of formalin diluted from 1 to 49 for
control measures. He leaves the corms in this solution for an hour,
without any damage being done to them. This proved to be a very
good way for controlling the English disease.

In the meanwhile, the formalin treatment has been applied at
one of the Calla-lily nurseries in Holland, and it was found, that
the disease was far less universal than before. It is hoped, that on
applying the method a second time, the disease will be entirely
got rid of.

Especially in the last few years many cases are reported of
cuttings which became infected by different species of Pythium.
In 1910 Peters (24) described the „Schwarzbeinigkeit" (Black-
leg) of geranium caused by P. de Baryanum. In 1922 van P o e-
teren (30) also reports the presence of P. de Baryanum on
geranium cuttings.

A publication on Blackleg of Pelargonium by W. B u d d i n
and E. Wakefield (9) appeared in 1923, in which P. de
Baryanum and Botrytis cinerea are named as the cause of the
disease.

In 1924, H. Pape (23) reported P. de Baryanum as a parasite
on carnation cuttings.

A detailed description of Pythium species occurring on geranium,
was published by Braun (8) in 1925. He describes two new
species, Pythium splendens and P. complectens.

In May 1925 some chrysanthemum cuttings, which had suddenly
stopped growing, were sent to me to be examined. It became evi-
dent, that the roots were suffering from a rot. I soon succeeded
in isolating a phycomycete from these decaying roots. This fungus
grew well on oatmeal-agar; it produced no oogonia however, but
a large number of conidia or swarmsporangia were formed in agar-
cultures. In order to be able to identify it, it was necessary to find
out in which way the zoospores were formed. I therefore placed

a tuft of the mycehum from agar-cultures in a watchglass with
water. It appeared, that this is not the right method for obtaining
zoospores. It was possible however to identify this fungus without
seeing swarmspores, when it is placed in water.

If, for instance, the mycelium in the watchglass was allowed
to stand for about twelve hours without the water being refreshed,
numerous sporangia were formed in chains. The first formed
specimen of such a chain often has a somewhat obovate shape, the
others are round. This corresponds with the description of Py-
thium intermedium given by de Bary (4). He also mentions
the absence of oogonia and antheridia. The chains of sporangia
are so characteristic, that it is nearly impossible to mistake it for
other Phycomycetes. De Bary found Pythium intermedium together
with Pythium de Baryanum in dead seedlings of Lepidium and
Amaranthus. Inoculation experiments however proved that it is
impossible for P. intermedium to penetrate into these seedlings
while they are living. But, according to de Bary it easily penetrates
into the tissues of healthy prothallia (Equisetum, Todea, Cera-
topteris). P. intermedium is also mentioned at length by Butler
in his publication on Pythium.

„It is curious, that this species, which is common as a
soil saprophyte, should have been so rarely observed. I
have so regularly obtained it from water, in which were
suspended fresh roots of Abutilon, that I at one time suspec-
ted parasitism on this plant. I however, failed to induce it
to attack living roots. De Bary equally failed to infect
Lepidium sativum and Amaranthus with it.

He, however, observed its parasitism on prothalli. At-
kinson found, that it was responsible for a disease of fern
prothaUi in the United States. The affected plants were soft,
limp and darker jn colour than healthy ones. A high tem-
perature, moist soil and air, and insufficient light and ven-
tilation, are the chief predisposing factors to damping-off.
When the soil is infected, it should be replaced by fresh

I also came across this species several times. From Louise Sol-
berg (Oslo) I received a strain of the same fungus, which she

had isolated from diseased tubercles of a lupin. It is therefore
probable, that P. intermedium often occurs in vegetable mould. It is
easily distinguished from the other Phycomycete-types with round
sporangia and without oogonia, which are so often found on de-
caying roots or in the soil, by the often long chains of sporangia,
which are formed when the mycelium is placed in water.

conidia of Pythium in- Inoculation experiments now had to prove,
termedium. 366 X P* intermedium is indeed able to infect

When I examined the infected plants two months afterwards
to study the course of the disease, it appeared that it was not of
a very serious nature. All plants, also those which had been most
badly infected, had outgrown the disease. This was probably also
due to more favourable conditions of temperature and humidity.

Up to now the presence of Pythium intermedium on chrysan-
themum or other cuttings has not been reported. I also wanted
to inoculate geranium cuttings with it, as they are apparently
easily attacked by different Pythium species. It is naturally thought,
that the fleshy, juicy stems of geraniums are more easily attacked
by Phycomycetes than the woody stems of chrysanthemums.

For comparison I also carried out some inoculation experiments
with Pythium de Baryanum isolated from peas, Pythium com-
plectens and P. splendens obtained respectively from Braun and
from Leonian and with a Phycomycete corresponding with P. de
Baryanum var. geranii Braun.

This last fungus is one of those forms without oogonia which
appeared when diseased roots were layed out on agarplates. It
is characterized by fairly large, dark, round conidia and many
chlamydospores of different sizes. I did not succeed in obtaining
swarmspores or oogonia from it.

The best results with inoculation-experiments were obtained
when the lower ends of the cuttings were rubbed with the myce-
lium from agar-cultures and then placed in pots with coarse steri-
lized sand. In order to get as much moisture as possible, the pots
were placed in socalled „sun-catchers", small glass houses built
by means of thick iron wires.

After a few days, the first symptoms of the disease appear.
The base of the cutting becomes black and the colour slowly
spreads towards the top. This phenomenon was also discussed
by Braun. The infection by Pythium splendens spreads through
the whole cutting, while that of P. complectens stops a few c.M.
from the base of the slip. I think that the typical dark black dis-
coloration of the tissues of geranium is characteristic for infec-
tion by Phycomycetes, while with Botrytis-infection a more brown
discoloration is typical. It appears, that on a whole geranium-
cuttings are more easily infected than chrysanthemum cuttings.

It only seldom happens that a chrysanthemum cutting is so badly
infected that it is killed at once. It is probable however, that the
somewhat diseased plants may show a temporary retardation in
growth.

As I always used fairly young slips I have not been able to
determine if the rot may also attack the roots, as one would suspect
when examining the originally diseased plants, which were much
older than the cuttings used by me. In any case, very special con-
ditions of temperature and humidity must appear, before such a
disease will be the result.

Inoculation experiments proved that Pythium intermedium is
strongly pathogenous for geranium cuttings, having the same
effect on them as P. splendens. Chrysanthemum cuttings on the
other hand, are only slightly attacked by it.

Pythium de Baryanum var. geranii, like P. de Baryanum proved
to be pathogenic for chrysanthemum cuttings, while P. splendens.
also attacked chrysanthemum slips.

1. Result of inoculation experiment in a hothouse with mean
temperature about 20°, on 11th. February \'26.

On 16th. February: 5 geraniums and 1 chrysanthemum
obviously infected, 1 geranium attacked at the base (this
one died afterwards).

On 18th. February: 1 chrysanthemum and 4 geraniums ob-
viously infected; 1 geranium black at the base, the others
healthy.

2. Result of inoculation experiment carried out in the same
hothouse on 23rd. April \'26.

12 cuttings of chrysanthemum and 6 cuttings of geranium
inoculated with P. intermedium.

On 29th. April: 4 geraniums turning black, 1 beginning to
show discoloration.
The other plants remain healthy.

3. Result of inoculation experiment carried out in the open in
sterilized sand on 17th. and 18th. June 1926. (See table p.
27). As Pythium intermedium often occurs in vegetable
mould, I presume, that blackleg of geranium is not only cau-
sed by Pythium de Baryanum, but also by P. intermedium. It
is however necessary to isolate the fungus from the disea-
sed geraniums in order to be able to say to what species
the blackleg is due.

In the last few years a great deal has been done in America
towards investigating the root rot of peas caused by Phycomy-
cetes. — Especially Fred. R. Jones did much in solving this
problem. In 1923 (16) he drew attention to the fact, that a fungus
which seemed to be a Phycomycete, is often found in the tissues
outside the endodermis of the roots of Legumes and some other
plants. He did not succeed in cultivating this organism outside
the roots. When it was present in large quantities it seemed to
cause a disease in the roots of Lathyrus, but under ordinary cir-
cumstances it did not appear to do much harm. Jones therefore
regards it as a kind of mycorrhizal fungus.

Perhaps it is a Phycomycete, but nothing further has become
known about it. F. R. Jones and his assistants however, after-
wards investigated other Phycomycetes which occur specially on
pearoots. A publication by Drechsler, Root rot of Peas in the
middle Atlantic States in 1924. appeared in 1925. (11)
The Phycomycetes which he isolated from pearoots are:

1. 6—8 species of Pythium of which 3 are usually included in
Pythium de Baryanum,

This last appeared to be the most pathogenic and is very
fully described by F. R. Jones and Drechsler (17). The

disease especially appears on fields on which peas have been
planted a few years in succession.

„Although some isolations were made from discoloured rootlets
not showing evidence of being attacked by any other fungus the
most prolific source for cultures of species of Pythium was found
in the cortical tissues of the stem and larger roots bearing the
oogonia and oospores of Aphanomyces euteiches. In fields in which
the latter fungus was common, the genus Pythium appeared to be
found occurring more abundantly in secondary relationships than
in directly parasitic ones. This condition is apparently not due
to any lack of potential virulence, since nearly all of the smooth
forms tried out so far, as well as one spiny form, which was deri-
ved from material collected at Hamburg, N. Y. attack cucumber
fruits with great readiness, generally a fair index of a moderate
degree of pathogenicity.

,,The widespread occurrence of even the most aggressively para-
sitic species of Pythium in dead organic matter has long been
recognized, and it is evident that the tissues killed by Aphano-
myces euteiches provide a more congenial substratum than the
living parts".

F. R. Jones and L i n f o r d published an account called:Pea
disease survey in Wisconsin, (18) in which the damage due to
species of Pythium is treated in one chapter.

„When pea plants suffering from root rot are examined in the
laboratory, the species of Pythium long known as a destructive
seedling parasite will often be found present in the diseased tis-
sue of many or all of the plants. Inoculation with some of the
cultures of Pythium obtained in this way has shown, that the
fungus is capable of preventing germination of pea seed or of
destroying many of the seedlings before they emerge from the
ground, and occasionally some degree of stem and root rot is
produced.

,,The study of the relation of Pythium to root and stem rot has
been greatly retarded by the fact, that the cultures obtained from
peas in the field have been found to belong to several species diffe-
ring somewhat in pathogenicity and frequency of occurrence and

furthermore it is not at all certain, that present methods used in
making isolations from plants secure cultures of all species present.
Thus a vast deal of work will be required before the relation of
Pythium species to stem and root injury of mature plants will be
fully known. It may be stated, however, that inoculations made
under controlled conditions have shown very slight ability in any
species studied thus far to produce either stem or root rot under
usual field conditions.

„During the summer, isolations were frequently made to secure
cultures of any species of Pythium, that might be present. Since
previous experience had shown, that some species are almost as
frequently found in association with roots apparently healthy as
with those diseased cultures were made from both. In making
cultures no sterilizing agents were applied to the surface of de-
caying tissue because they penetrate rapidly and destroy all Phy-
comycetous mycelium quickly. Thus mycelium adhering to the
outside of roots may give fise to a culture, a difficulty which can
hardly be avoided.

„This table corroborates previous experience that cultures of
Pythium are obtained with approximately the same frequency
from healthy as from diseased plants. It also shows that in
about one-third of the isolations two species were obtained, though
in such cases no two species seem to be found more frequently
associated than others.

,,From the observations made thus far it appears that some of
these species of Pythium occurring abundantly in the soil may at
times be responsible for the death of root ends and for some rootlet
injury, but only rarely for root and stemrot as it is usually known.
It is possible that almost universal invasion of the root cortex of

peas and clover by a mycorrhizal fungus renders this tissue espe-
cially accessible to these species and that it is from this super-
ficial invasion that the fungus is most frequently obtained in cul-
ture". —

In Europe, Pythium Sadebeckianum is reported occurring on
peas. Butler (10), however, omits this species. He remarks about
it: ,,This species occurred as a parasite on peas in Pomerania and
lupines in Hamburg, doing a considerable amount of damage in
1891. The diseased peas were examined by Wittmack, who pu-
blished a brief account of the pathological conditions without, ho-
wever, any diagnosis of the fungus further than the measurements
of bodies which resembled the oospores of a Pythium. Sadebeck,
who saw the specimens considered it to be a new Pythium, allied
to P. de Baryanum and P. Equiseti. The oogonia measured 32
H in diameter. Whether it is the same as the species generally in-
cluded under P. de Baryanum, which has been reported attacking
peas in the United States, England and elsewhere is uncertain".

These are the most important publications which have appeared
about the rootrot of peas caused by Phycomycetes.

In order to determine whether Aphanomyces euteiches is also
found in this country, I examined several fields on which peas
were cultivated. It was done in the first part of May as the plants
are often attacked early in the season. Although a lot of rain fell
in the spring of 1926 and conditions were therefore favourable
for the development of the disease, I only found a few plants
showing signs of disease. Only on one field a few rows of plants
were apparendy affected.

The affected plants first became yellow or mottled and then
dried out. If the plants were taken out of the soil it could be ob-
served that the roots showed signs of decay. I collected diseased
plants from different parts and examined them microscopically.
In the roots I found hyphae and sometimes oogonia belonging to
some kind of Phycomycete, but I never met the peculiar hyphae
of the mycorrhizal fungus described by Jones.

In isolating the fungus from diseased roots I of course had to
cope with the same difficulties as Jones.

sterilized, one never knows if an isolated species is actually the
same one as that of which oogonia appear in the roots. Only
inoculation experiments could prove if a certain isolated species
would be pathogenic or not.

From most of the isolations a species was obtained with oogonia
often possessing protuberances. I had met this fungus before in a
young lupin which had shown the typical symptoms of damping-off.

When a colleague tried to isolate Cladosporium cucumerinum
from cucumber seeds which failed to germinate the same species
appeared in the isolations. Apparently it is a common soil fungus.
Except this fungus with irregular oogonia, which I usually ob-
tained from diseased roots, other forms appeared without oogonia
and which were therefore hard to identify. I have however chiefly
confined myself to the fungus with irregular oogonia which I shall
afterwards describe as Pythium irreguläre n. sp.

Besides I carried out some inoculation experiments on peas with
one of the fungi without oogonia, which corresponds with P. de
Baryanum var. geranii Braun. Although this fungus had not been
isolated from peas it had appeared to be very virulent during
experiments on cuttings. For comparison I also carried out some
inoculation experiments with Pythium de Baryanum and Aphano-
myces euteiches.

The former I had isolated from diseased pea roots, the latter
was obtained as a pure culture from the United States (Drechsler).

For my first inoculation experiments I used humus, through
which I mixed agarcultures of the fungi before sowing the peas
in it. At the time I had not yet isolated Pythium irreguläre from
pea roots, so that I mainly used Pythium de Baryanum and P. de
Baryanum var. geranii. It soon became evident that this method
is not very satisfactory. The little radical was immediately at-
tacked by the fungus and the peas did not come up. If they did,
they either were very small and miserable or they outgrew the
disease, but in neither case did they show the typical symptoms
of root rot. Another drawback was that I had to use peas with a
high percentage of germination, as it would otherwise be impos-

sible to carry out control experiments. „Wonder van Amerika",
the variety I used, had a very high germinative power, but on the
other hand it is more resistant to disease than other varieties of
green peas (doperwten). When working with less virulent para-
sites there would therefore be more chance of the inoculation suc-
ceeding with less resistant varieties of green peas, than with the
Wonder van Amerika. I arranged the inoculation experiments in
the following way: the peas were sown in pots containing steri-
lized vegetable mould mixed with oatmeal-agar cultures of the
different fungi in question.

These difficulties could be removed by using watercultures. One
then also has a better survey of the progress of the rot. Other
soil fungi which in pot experiments always follow on the Phyco-
mycetes were eliminated in this way. The roots which have grown
in watercultures are clean and therefore very suitable for micros-
copical examination.

The seeds were allowed to germinate and the best seedlings
were then sorted out and used for the waterculture-experiments.

It possesses long, thin roots, while seedlings of Wonder van
Amerika have a short firm root system.

I proceeded as follows: The seeds were first sterilized with
alcohol-sublimate (1 alcohol 96 % : 1 sublimate 0,1 % to prevent
infection in the germinating dishes as far as possible.

They were then laid in dishes on wet filterpaper and when the
roots were a few c.M. long they were used for the watercultures.

In the lid of a jampot three holes were made and in these holes
two pieces of cork, somewhat hollowed out in the middle, were
fitted. The roots of the seedlings were pushed through the round
hole, which was formed between the two pieces of cork, while
the cotyledons rested on the top of them.

After the roots had grown in the solution for a little while, I pla-
ced a tuft of the mycelium from the fungus with which I wanted to
inoculate, against them. It may of course be objected against this
method that the fungus has an advantage over the plant in this
way. A pea is not a waterplant and is therefore not cultivated
under normal circumstances, when growing in watercultures.

Phycomycetes on the other hand are water-fungi and easily pro-
duce swarmspores in mineral solutions, so that in watercultures
the circumstances are very favourable for infection. The pea seed-
lings, however, grow so well in v. d. Crone\'s culture solution, and
produce such a healthy root system, that according to my opinion,
they may well be compared with plants grown under normal cir-
cumstances. At the utmost one may get an exaggerated idea of
the pathogenicity of the parasite, when one relies on the damage
done by it to the root system of pea seedlings in watercultures.

But then the damage done to the green parts of the plant is
usually less striking in watercultures than on the fields. On the
fields the root rot is characterized by the drying out of the in-
fected plants and this of course does not soon happen in water-
cultures: as long as a few roots remain intact the plant will not soon
dry out. Although the whole aspect of the disease is therefore
somewhat different in watercultures than in the pots, one may
still draw conclusions about the pathogenicity of a special Phy-
comycete for the roots of peas. Aphanomyces euteiches appeared

to be the most virulent of the four Phycomycetes (Aphanomyces
euteiches, Pythium de Baryanum, P. de Baryanum var. geranii
and Pythium irreguläre), with which inoculation experiments were
done.

A little while after the pea roots had been inoculated with Apha-
nomyces euteiches the whole root system became limp.

They did not become as glassy and transparent as with Pythium
inoculations but remained white. The infection is very acute. Even
in watercultures the plants may shrivel up and die.

When inoculated with Pythium irreguläre the roots were in many
cases also soon badly infected. On the upper part of the plants no-
thing could be seen however. The growth was normal and the leaves
were dark green. The roots were glassy, transparent and brown
in colour, while there were only a few small side roots at the
ends. These side roots may, when they are also infected, show
a reddish brown ring at the place where they are attached to the
main roots. When roots infected by Aphanomyces euteiches or
Pythium irreguläre are examined microscopically, large numbers
of oogonia are found in them.

Pythium de Baryanum and P. de Baryanum var. geranii also
attack pea roots in watercultures. In their method of infection they
resemble Pythium irreguläre and not Aphanomyces euteiches.

The plants in the control experiments remained healthy and
possessed a clean white, well branched root system.

From these results I think I may conclude that Pythium irregu-
läre is the cause of a root rot of peas, and that a large number
of the diseased plants with decaying roots, which I found on the
fields, is due to it. Finally it may be pointed out, that it is hard
to distinguish Pythium irreguläre from Pythium de Baryanum when
the infected roots are examined microscopically.

The protuberances of the oogonia are very hard to find in the
tissues of the roots.

Many of the isolations which I made of this Pythium showed
different variations. I specially kept three of them in culture, one

from peas, one from lupins, one from cucumber seeds. Macro-
scopically these fungi look exactly like other Pythiums. I usually
cultivated them on oatmeal-agar on which they made the usual
white woolly growth of mycelium. I tried to get swarmspores by
the method of Petri. The strain isolated from cucumber seeds
produces a few swarmspores in this way, but there were too few
to observe the discharge of the swarmspores from the sporangia,
or to be able to count the cilia. For the purpose of counting the
cilia, I placed a drop of water with swarmspores on a slide with a
drop of eosine-sublimate (following the advice of Mr. v. Luyk).

The swarmspores were immediately fixated and coloured by
the eosine before they withdrew the cilia. It is obvious that one
can only apply this method when there are a fairly large number
of swarmspores at one\'s disposal. It was impossible for me to
count the cilia, as the strain from cucumber only produced a few,
the strain from lupin only very few single ones, and the strain
from pea no swarmspores at all.

As I could not determine the
way in which the swarmspores
were formed the difficulty was to

the genus Pythium, owing to the great similarity which exists
between this fungus and P. de Baryanum.

The only difference which really exists between this species and
P. de Baryanum is the irregular shape of the oogonia. The oogonia
often (but not always) carry protuberances, varying in size and

shape, and sometimes separated from the oogonium by a trans-
verse wall. Thesq protuberances are especially found in cultures

a few weeks old. The oogonia are mosdy intercalary and the
oospores, sometimes two in number, lie free in the oogonium. The
antheridia are usually of the same type as those of P. de Baryanum
but I never saw hypogynal antheridia. One oogonium is sometimes
surrounded by more antheridia. The conidia or swarmsporangia
are globose or somewhat obovate, and differ in size and shape.

I think that this fungus is sufficiently distinguished from P. de
Baryanum by the protuberances found on the oogonia, to justify
recording it as a new species for which I propose the name Pythium
irreguläre.

Pythium irreguläre nov. sp.: Oogonia often with protuberances
of different sizes and shapes.

Oogonia sometimes globose, sometimes irregular in shape. When
globose, often i le—18 ^ in diameter, usually intercalary.
Oospore free in the oogonium, ordinarily 14—16 ^ in diameter.
The wall of the oospore has a thickness of about 1 — 1\'/a /x.

Antheridia produced on the same hyphae as the oogonium,
sometimes several round one oogonium. Swarmsporangia and coni-
dia vary greatly in size, ± 10—20/i, globose or obovate. Swarm-
spores seldom produced, 8 /z in diameter when in rest. Iso-
lated from pea roots, lupin plant and cucumber seeds. Distinguished
from P. de Baryanum by the protuberances found on the oogonia.

When examining pea roots microscopically I sometimes obser-
ved Asterocystis radicis d e W i 1 d e m a n (37) in the parenchy-
mal cells. This fungus belongs to the Chytridiaceae and Mar-
chai (21) described it as being the cause of the Belgian and
Dutch „vlasbrand". a name, which we might translate by „flax-
fire". It is a typical wilt-disease, not identical however with the
Fusarium flax wilt of the U. S.

In Holland the „vlasbrand" occurs principally in the northern
provinces, Groningen and Friesland. When the disease is very
acute large open spaces appear in the fields. The flax plants in
these places are small, the leaflets wilt, they turn yellow and
shrivel up. The plants look as if they were scorched by fire. Under
favourable conditions the plants sometimes outgrow the disease,
but they never quite recover.

The blue flax is much more susceptible than the white variety.
In severe epidemics however, as in 1926, the white flax is also
attacked. On old heavy clay soils, the „brand" rarely occurs, but
on the lighter sandy clay, the disease is very common. It is remar-
kable that even in the new „polders", where flax is grown for
the first time, the „brand" may occur. This disease is of a severe
nature; the yield on those fields is extremely small. D r. Z ij 1 s t r a,
chief of the botanical department of the Groningen Experiment
Station, called it „a pest for our best flax regions".

Marchal found Asterocystis radicis in all the „brand" speci-
mens from different parts of Belgium. He proved the parasitic
nature of this fungus by carrying out inoculation experiments. He
grew flax in watercultures and inoculated the nutrient solution
with Asterocystis; the result was a thorough infection of the
roots. By growing the plants in infected soil, he also obtained
diseased plants. The presence of Asterocystis in the roots is easily
ascertained by a simple staining process with potassium-iodide.
Marchal then determined that the chlamydospores and swarm-
sporangia were stained a deep brown-red. Where the symptoms
of the disease were concerned Marchal remarks: „The less water
the plants have at their disposal, the stronger they react on the
lack of water, which becomes evident after infection and the more
striking are the symptoms of the „brand" ".

The moisture, which by influencing the dissemination of the
swarmspores helps to spread the disease, on the other hand dimi-
nishes its pernicious influence.

Marchal\'s investigations are the only ones published about this
disease. Dr. Zijlstra informed me how to obtain infected plants
in spring. He therefore sent me a sample from „brand" soil,
which had been collected two and a half years ago. He advised
me to grow flax in nutrient solutions (van der Crone). The bot-
tom of the jar was covered with a thin layer of the soil. This in-
fection succeeded beautifully. A large part of the root-cells was
filled up with the Asterocystis sporangia. The plants of course,
having their roots in the liquid, did not shrivel like those in the
field, although they had a sickly appearance. The control plants
were quite healthy.

When growing peas in the same solution, I also noticed a slight
infection of the roots. In this case however the plants remained
quite healthy.

In the summer of 1926 I visited the flax district of Groningen
and collected plants of a „brand" spot in the field. I was there told
by one of the foremost farmers, that these plants were typical
„brand" plants.

The examination of these specimens however revealed relatively
few sporangia of Asterocystis, but a great many round spiny
bodies, which struck me as being oogonia of a Phycomycete. Later

in the summer I had the opportunity of examining flax roots from
Friesland, and again I noticed the same phenomenon; the spores
of Asterocystis however were more abundant in the roots here.

The question now arose, whether the root rot of flax might be
caused by the Phycomycete in question, as well as by Astero-
cystis radicis. It would not be surprising if the wilting and shrivel-
ing of the plants were caused by different parasites. The „vlas-
brand" would then be a complex of diseases.

It is possible that several kinds of root parasites might nearly
have the same effect. We know f. i. that Thielavia basicola may
cause a rootrot of flax. —

The Phycomycete proved to be Pythium megalacanthum d e
Bary (4), which was isolated very easily from the decaying
roots. I thus intended to make inoculation experiments on the roots,
with the exclusion of Asterocystis. For this purpose the flax seeds
were germinated on wet filter-paper. The young plants were
put in the nutrient solution and mycelium from the pure cultures
on oatmeal-agar was smeared on the rootlets.

During the first days after the inoculation the roots apparently
develop normally. After some days however, the top of the prin-
cipal root begins to soften. This „rotten" end is separated from

Except the main root, the lateral roots are also attacked. All these
infected roots become glassy and limp.

Fig. 12 shows a piece of flax root from such an artificially in-
fected waterculture. The spiny oogonia appear in large numbers
in the roots.

It therefore appears that it is possible to cause a rot of the
flax roots by Pythium megalacanthum, even when Asterocystis
radicis is absent.

As Marchal already noticed with his experiments with Astero-
cystis, the plants in watercultures do not suffer very much from
wilting. This also counts for Pythium megalacanthum. The aerial
parts of the plants do not shrivel up.

It was only my intention to focus the interest on the fact that
„vlasbrand" is not such a simple disease, as one would like to
imagine.

Before methods of control may be found, it.will be necessary
to analyse the different factors of this complex-phenomenon.

The large spiny oogonia of the Phycomycete which I found
in flax roots, naturally reminded me of the illustrations of the
oogonia of Pythium megalacanthum given by d e B a r y. De Bary
found Pythium megalacanthum in seedlings of garden-cress which
had already been attacked by Pythium de Baryanum. He tried
to infect the seedlings by inoculating them with Pythium megala-
canthum but did not succeed. He then inoculated prothallia of
the fern Todea africana with it and some of them were attacked.
He always used zoospores for his inoculations. According to Butler
this fungus has never been described again except on one occa-
sion when Schröter states, that he believed he found it in the
stems of Veronica hederaefolia.

The Phycomycete which I isolated from flax seemed to corres-
pond very well with the description given by de Bary for P. mega-
lacanthum.* The oogonia were often larger in size than the

measurements given by de Bary, but they vary so much that
this is of no importance. De Bary already pointed out that the
spores of P. megalacanthum vary more in size than those of P.
intermedium. The drawing of the infected flax root (fig. 12)
shows the variations in size of the oogonia, which range from
± 30 to 70 At. I did not succeed in getting zoosporangia. Amongst
his different cultures on seedlings of garden-cress, de Bary also
had one form which possessed numerous oogonia and only few
swarmsporangia. Unfortunately I only possess one isolation and
perhaps that is the reason for the absence of the swarmsporangia.
Even the method of Petri gave no results. P. megalacanthum
grows on oatmeal- and on cornmeal-agar but not on cherry-agar.

The cultures have a granular appearance owing to the many
large oogonia. Many oogonia are quite empty and the contents
have degenerated. This was also noticed by de Bary who takes

In connection with the inoculation experiments, it was neces-
sary to know whether many Phycomycetes appear in vegetable
mould in which the various diseased plants, which I had examined,
grew. If many Phycomycetes occurred, the experiments with un-
sterilized soil would be worthless. With my first experiment in
which the peas were sown in unsterilized soil, I already noticed
that an infection by Pythium de Baryanum appeared in the con-
trol experiment. Unsterilized soil could therefore not be used.

When a decaying root is laid in a petridish on cherry- or on
oatmeal-agar several Pythiums often start growing out of it. In
the chapter about the root rot of peas I already pointed out that
according to Drechsler (11) this may happen even if the
rot is caused by a different parasite (in that case by Aphanomyces
euteiches). F. R. Jones and Lin ford (18) even isolated
several Pythiums from healthy pea roots.

One may gain an impression of the great number of Phycomy-
cetes occurring in vegetable mould by laying out roots of different
plants.

The many types which appear, make a research about Phyco-
mycetes rather difficult at first.

One comes across several forms without oogonia or without
swarmsporangia. With such isolations one often meets Pythium
de Baryanum and the form called P. de Baryanum var. geranii
by Braun. These forms were also often isolated from various
seedlings.

inum from cucumber seeds which failed to germinate in vegetable
mould, but on one occasion he obtained different Pythiums in-
stead. He handed them over to me and it appeared that Pythium
intermedium and Pythium irreguläre were present. From these
seeds I also isolated a form with dark, somewhat spiny oogonia.
I afterwards never came across this form again. Although the
seeds were put to germinate in a hothouse, the temperature could
hardly be the cause of the occurrence of this form, as many of
my own seedlings were also cultivated at about the same tem-
perature.

From decaying roots of pansies which had been grown m ano-
ther soil-sample at a lower temperature, I isolated an Aphano-
myces which looked very similar to Aphanomyces euteiches, but
which, in my infection experiments, did not attack pea roots. Un-
luckily I did not have time to carry out inoculation experiments
with it on pansies, and I have therefore not been able to ascertain
whether this Aphanomyces is the cause of the acute root rot of
pansies appearing in our experimental garden. From Calla-hly-
roots I isolated, besides Phytophthora Richardiae, a few other
forms,probably also Pythiums.

I never obtained Phytophthora species in the same way and ap-
parently they do not lead the same saprophytic life in the soil as
the different species of Pythium. The question of the temperature,
which may be of importance here, was not investigated however. —

It is evident that vegetable mould is full of different Phycomy-
cetes and that inoculation experiments with unsterilized humus

these isolations was that the dishes were often overgrown by a
Mucor. But here again other forms than those which I had isola-
ted from vegetable mould appeared.

1. It has become evident that there are no sound reasons for
regarding Pythiomorpha Petersen and Blepharospora
Petri as constituting new genera. There is no objection
against including them in the genus Phytophthora.

In practice it is not desirable that the genera Pythium and
Phytophthora should be placed in one genus, although previ-
ous investigators have shown that theoretically there is no
difference between them.

2. Inoculation experiments proved that a serious root rot of Cal-
la-lilies, which causes a considerable damage in the nurseries,
is caused by a Phytophthora species. As this species is not
identical with any of the ones previously described, the name
Phytophthora Richardiae n. sp. is proposed.

A treatment of the Calla corms with formalin, which has
already yielded good results in practice, is given as a control
measure.

3. It appeared that Pythium intermedium d e Bary, isolated
from chrysanthemum cuttings, which had stopped growing,
is strongly pathogenic for geranium cuttings and may also
attack chrysanthemum cuttings. Inoculation experiments were
also undertaken with geranium cuttings, as a publication by
Braun about the rot of these slips appeared in the United
States.

For comparison I also inoculated with the species P. splen-
dens and P. complectens named by Braun. These species,
which had been kept in culture at the Centraal Bureau voor
Schimmelcultures for some time, were still very virulent.
P. splendens also attacked chrysanthemum cuttings. Inocula-

tion experiments with the strains of P. de Baryanum and
P. de Baryanum var. geranii which I isolated, gave positive
results with geranium and chrysanthemum cuttings.

4. A Pythium with irregular oogonia sometimes with protube-
rances, was isolated from the roots of a large number of the
pea plants I found suffering from root rot.

As far as I know this species has not been described before
and I therefore give the description and propose the name

Inoculation experiments in watercultures proved that it
attacks the roots of peas and is the cause of a root rot. I
never came acrose Aphanomyces euteiches in peas, but in-
oculation experiments showed that it is more virulent than

5. Typical „vlasbrand" has been ascribed to Asterocystis radi-
cis de Wildeman.

something is the matter with the roots. When examining the
diseased plants I often found Pythium megalacanthum de
Bary present in the roots. Inoculation experiments showed
that this species attacks the roots of flax. As I found this
species in the roots of diseased plants from different districts
I think I may conclude that like Asterocystis radicis it may
be one of the causes of the disease. A thorough re-investi-
gation of vlasbrand is however necessary.

6. Inoculation experiments with Phycomycetes on roots are
very much simplified by using watercultures.

7 It appeared that various Phycomycetes may be isolated from
decaying parts of plants grown in vegetable mould. These
forms are often very hard to identify owing to absence of
oogonia or sporangia.

3. A. DE BARY, Untersuchung en über die Peronosporeen und
Saprolegnieen und die Grundlagen eines natürlichen Systems
der Pilze.

1921, Seventh Annual Report, Exp. and Research Station,
Turner\'s Hill, Cheshunt.

8. H. BRAUN, Comparative studies of Pythium debaryanum
and two related species from geranium.

1924, Annals of the Royal Botanic Gardens. Peradeniya,
\' Vol. IX. Part. 1, June.

16. F. R. JONES, Mycorrhizal fungi in the Roots of Legumes
and some other Plants.

17. F. R. JONES and C. DRECHSLER. Root rot of Peas in the
United States caused by Aphanomyces euteiches n. sp.

1925, Agr. Exp. Station of the Univ. of Wisconsm, Ma-
dison, Research Bulletin 64.

22. M. VON MINDEN, Beiträge zur Biologie und Systematik
einheimischer submerser Phycomyceten.

23. H. PAPE, lieber eine durch Pythium de Baryanum Hesse
verursachte Stecklingskrankheit der Nelken.

25. HENNING E. PETERSEN, An account of Danish Fresh-
water-Phycomycetes with biological and systematical remarks.

26. GEO. H. PETHYBRIDGE and H. A. LAFFERTY, A disease
of tomato and other plants caused by a new species of Phy-
tophthora.

29. L. PETRI, Nuove osservazioni sulla biologia e sul parassi-
tismo della Blepharospora cambivora.

30. N. v. POETEREN, Verslag over de Werkzaamheden van
den Plantenziektenkundigen Dienst in de jaren 1920 en 1921.

31. R. D. RANDS, Streepkanker van kaneel, veroorzaakt door
Phytophthora Cinnamomi n. sp.

35. R. E. SMITH and E. H. SMITH, Further studies of Pythia-
ceous infection of deciduous fruit trees in California.

1904. U. S. Department of Agriculture, Bureau of Plant
Industry, Bulletin no. 60.
37. E. DE WILDEMAN, Asterocystis radicis n. sp.

§ 1. The classification in the Phytophthoreae
§ 2. The classification in the genera Phytophthora and

tion of the pathogenic organism
§ 3. The pathogenic organism
§ 4. Inoculation experiments

§ 2. Inoculation experiments
§ 3. Description of Pythium irreguläre n. sp. in culture

7
10

14
16

22
24

28
32
35

39
42

44
46
48

" \' ■ - \' ■■ ^ \' : - ■ . ■ ■ ■
\'\' \' ■■ - \' ^V- \' / .\'cj\'- . - .,.

De meerdere of mindere resistentie van vlasrassen tegen infectie
door Fusarium lini berust op het voorkomen bij die rassen van
een grootere of kleinere hoeveelheid glucoside.

De pseudo-vacuole der blauwwieren. die voornamelijk optreedt
bij soorten, welke waterbloei veroorzaken, heeft een protoplasma-
tischen inhoud.

De verdeeling der water-organismen in saprophielen, saprotole-
ranten en obligaat katharoben verdient de voorkeur boven die in
saproben en katharoben.

Noord-Holland ten Noorden van het Noordzee-kanaal weer bevolkt
wordt met typische zoetwater-organismen, die er thans niet voor-

	Number of ino- culated plants	Heavily affected	The infection stops on some distance from the base	Healthy
P- splendens	6 ger.; 6 chrys.	6 ger.; 1 chrys.	2 chrys.	3 chrys.
P- complectens	3 ger.; 3 chrys.		3 ger.	3 chrys.
P. intermedium	6 ger.; 6 chrys.	6 ger.	2 chrys.	4 chrys.
P. de Baryanum	6 ger.; 6 chrys.	4 ger.	2 ger.; 2 chrys.	4 chrys.
P. de B. var. geranii	6 ger.; 6 chrys.	5 ger.	1 ger.; 2 chrys.	4 chrys.
Control	5 ger.; 5 chrys.			5 ger.; 5 chrys.

	Number of seeds	Number of plants living after 11 days
Inoculated with:
Pythium de Baryanum (isolated from pea-roots)	15	0
Pythium de Baryanum (strain from Hardey)	15	1
Pythium de Baryanum (Strain from diseased corm of Gloxinia)	6	1 poor specimen
Pythium de Baryanum var. geranii	15	3 (one of which very small)
Control	5	5

	No culture of	1 species of	2 species of
	Pythium obtained	Pythium obtained	Pythium obtained
Healthy pea roots	10	22	H
Decaying pea roots and stems	6	16	8